Wasp-protecting small molecules, compositions, methods and uses thereof in the treatment of innate and acquired immune-related disorders or conditions

ABSTRACT

The present invention provides specific small molecule compounds that modulate degradation and stability of Wiskott-Aldrich Syndrome protein (WASp), methods and uses thereof in innate and acquired immune-related disorders or conditions, specifically, in primary and secondary immune-deficiencies.

FIELD OF THE INVENTION

The present invention pertains to the field of molecular immunology andhematology. More specifically, the present invention provides specificsmall molecule compounds that modulate degradation of Wiskott-AldrichSyndrome protein (WASp), methods and uses thereof in innate and acquiredimmune-related disorders or conditions.

BACKGROUND ART

References considered to be relevant as background to the presentlydisclosed subject matter are listed below:

-   H. D. Ochs, A. J. Thrasher, The Wiskott-Aldrich syndrome. The    Journal of Allergy and Clinical Immunology 117, 725-738; quiz 739    (2006).-   M. H. Albert, et al Blood 115, 3231-3238 (2010).-   J. M. Derry, et al., Cell 79, following 922 (1994).-   O. Matalon, et al., Immunological Reviews 256, 10-29 (2013).-   N. Joseph, Biochimica et Biophysica Acta (BBA)—Biomembranes,    105:481-93 (2013).-   Y. Jin, et al., Blood 104, 4010-4019 (2004).-   M. I. Lutskiy, F et al., Journal of Immunology 175, 1329-1336    (2005).-   D. Buchbinder, et al., The Application of Clinical Genetics 7, 55-66    (2014). Filipovich, Bone Marrow Transplantation 42 Suppl 1, S49-S52    (2008).-   C. R. Shin, et al., Bone Marrow Transplantation 47, 1428-1435    (2012).-   J. Litzman, et al., Archives of Disease in Childhood 75, 436-439    (1996). L. Khavrutskii, et al., Journal of Visualized Experiments:    JoVE 15, (2013) A. Konno, et al., International Immunology 19,    185-192 (2007).-   E. Noy, et al., International Journal of Molecular Sciences 13,    7629-7647 (2012).-   S. Fried, et al., Science Signaling 7, ra60 (2014).-   B. Reicher, et al., Molecular and Cellular Biology 32, 3153-3163    (2012)-   M. Barda-Saad, et al., Nature Immunology 6, 80-89 (2005) M. I.    Lutskiy, et al., British Journal of Haematology 139, 98-105 (2007)-   A. Shcherbina, et al., Blood 98, 2988-2991 (2001)-   J. K. Burkhardt, et al., Annual Review of Immunology 26, 233-259    (2008).-   D. Varga-Szabo, et al., Journal of Thrombosis and Haemostasis 7,    1057-1066 (2009).-   Aiuti, L. et al., Science 341, 1233151 (2013).-   S. Hacein-Bey Abina, et al., JAMA 313, 1550-1563 (2015).-   H. Albert, et al., Science translational medicine 6, 227ra233    (2014).-   F. C. Peterson, et al., The Journal of biological chemistry 282,    8446-8453 (2007).-   M. H. Pauker, et al., Molecular and cellular biology 31, 2653-2666    (2011).-   H. M. Pauker, et al., Science Signaling 5(221):rs3 (2012)-   F. A. Ran, et al., Nature protocols 8, 2281-2308 (2013).-   S. B. Snapper, et al., Immunity 9, 81-91 (1998).-   J. A. Doudna, et al., Science 346, 1258096 (2014).-   Charrier, S., et al., Gene therapy 14 (2007), 415-428.-   Snapper, S. B., et al., Journal of leukocyte biology 77 (2005),    993-998.-   Zhang, H., et al., Immunity 25 (2006), 285-295.-   Gunn, M. D., et al., The Journal of experimental medicine 189    (1999), 451-460.-   Altman, L. C., et al., The Journal of clinical investigation 54    (1974), 486-493.-   Linder, S. & Kopp, P. Journal of cell science 118 (2005), 2079-2082.-   Linder, S., et al., Proceedings of the National Academy of Sciences    of the United States of America 96 (1999), 9648-9653.-   Zhang, J., et al., The Journal of experimental medicine 190 (1999),    1329-1342.-   Matalon, O., et al., Science signaling 9 (2016), ra54.-   Lee, S. H., et al., J Immunol 183 (2009), 7931-7938.-   Anfossi, N., et al., Immunity 25 (2006), 331-342.-   D. Schneidman-Duhovny, et al. Nucleic acids research 33, W363-367    (2005).

Acknowledgement of the above references herein is not to be inferred asmeaning that these are in any way relevant to the patentability of thepresently disclosed subject matter.

BACKGROUND OF THE INVENTION

Wiskott-Aldrich syndrome (WAS) is a severe primary immunodeficiencywhose symptoms include recurrent infections, severe bleedings caused bymicrothrombocytopenia; a decrease in the number and size of platelets,and an increased incidence of autoimmunity and lymphoma (Ochs et al,2006). X-linked thrombocytopenia (XLT) is considered a “mild” version ofWAS, and is characterized mainly by frequent bleeding events caused bythe severe microthrombocytopenia of these patients (Albert et al 2010).The defective gene responsible for WAS or XLT, the WAS gene, is locatedon the short arm of the X chromosome (Xp11.22-p11.23.1) and encodes a502-amino acid protein- the WAS protein (WASp) (Derry et al, 1994).

WASp, which is expressed in hematopoietic cells, is an adaptor proteinthat facilitates actin cytoskeletal rearrangements, which are essentialfor normal immune cell responses. WASp-dependent immune cell functionsinclude actin polymerization, sustaining of the immunological synapse,endocytosis, calcium flux, NFAT gene transcription, cellular activationand proliferation (Matalon et al, 2013; Joseph et al, 2013). More than300 mutations spanning the entire WAS gene have been identified. Thesemutations reduce WASp expression, which is strongly correlated with theseverity of the disease. The severe phenotypes manifested in classicalWAS usually occur where WASp is completely absent, while partial orresidual WASp expression is associated mainly with the less severephenotype of XLT (Jin et al, 2004; Lutskiy et al, 2005). WAS and XLT arediagnosed in about 1 to 10 patients per million male newborns worldwide.Without early intervention, most WAS patients die during the firstdecade of their life from opportunist infections, while XLT patientsexhibit a normal life span but frequently suffer from life-threateninghemorrhages (Ochs et al, 2006; Buchbinder et al, 2014).

Intriguingly, WAS gene mutations are usually normally transcribed, andaberrant WASp expression is therefore the result of post-translationalprotein instability. Accordingly, the majority of WAS mutations alterWASp binding to WASp-interacting protein (WIP), a chaperone of WASp thatprotects WASp from proteolysis (Reicher et al, 2012; Lutskiy et al,2005; Konno et al 2007; Noy et al, 2012). The molecular degradationmechanism of WASp was recently deciphered by the present inventors(Reicher et al, 2012; Fried et al, 2014). In resting cells WASp is autoinhibited and is tightly bound to WIP, which masks its degradationsites, lysine residues 76 and 81, located in a pocket at the N′-terminalWASp-homology-1 (WH1) domain of WASp. Following cellular activation acascade of signaling events leads to the release of WASp from autoinhibition, its recruitment to the cell membrane and actin rearrangementat the leading edge. Later in the activation process, WASp isphosphorylated on tyrosine 291 and WIP is phosphorylated by PKCθ onserine 488. These phosphorylation events induce the recruitment ofCbl-family E3 ligases to WASp, and trigger a conformational change thatreleases WIP protection from the degradation pocket of WASp.Subsequently, WASp lysines 76 and 81 are ubiquitylated, marking it forproteasomal degradation. Thus, partial or complete WASp deficiency ofWAS/XLT patients is the result of constant, unregulated, ubiquitylationand degradation of WASp that is not properly protected by WIP.

Unfortunately, currently there are no curative treatments for WAS/XLT,except for allogeneic hematopoietic stem cell transplantation (HSCT),which requires identification of a suitable donor and can result insignificant complications (Filipovich et al, 2008; Shin et al, 2012).Moreover, due to high risk-benefit ratio, HSCT is usually not arecommended treatment for XLT patients. For these patients, electivesplenectomy is usually the treatment of choice, as removal of the spleenhas shown to increase platelet count. However, splenectomy is notcurative and has potential risks, such as insufficient production ofantibodies and infections (Albert et al 2010; Litzman et al, 1996).Therefore, a safe and effective treatment is needed, which addresses theneeds of both WAS and XLT patients.

WO 2014/195857 that is a previous publication by the inventors,discloses liposomal compositions comprising WASp modulators that may beused either in reducing WASp levels in hematopoietic malignancies, oralternatively, enhance WASp levels in WAS, XLT and associated disorders.

As WASp is a key regulator essential for the activity and function ofmost immune cells, increasing WASp expression offers a method topotentially boost the performance of the immune response.

The need for a safe and efficient treatment for both WAS and XLT led theinventors to develop a new and different therapeutic approach, whichunlike gene correction, is based on the molecular mechanism of thedisease and focuses on defending native WASp from degradation ratherthan replacing it.

SUMMARY OF THE INVENTION

A first aspect of the invention relates to an effective amount of atleast one small molecule compound (SMC) modulator or any vehicle,matrix, nano- or micro-particle comprising the same, for use in a methodfor modulating the degradation and stability of Wiskott-Aldrich Syndromeprotein (WASp) in a cell. In more specific embodiments, the modulator ofthe invention may have the general formula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other to be absent orselected to be absent or from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—,—(CH₂)—, —(CH₂)—O—, —NH—(CH₂)—, and each optionally substituted withC₁-C₅ alkyl, a ring system containing five to twelve atoms optionallysubstituted with C₁-C₅ alkyl;

R₃ and R4 are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl.

A further aspect of the invention relates to a method for modulatingdegradation and/or stability of WASp in a cell. More specifically, themethod comprises the step of contacting the cell with an effectiveamount of at least one of the SMC modulators of WASp as described by theinvention or a pharmaceutically acceptable salt, esters or hydratethereof or any analogs or derivatives thereof, any combination thereof,or any vehicle, matrix, nano- or micro-particle, or compositioncomprising the same.

In yet a further aspect, the invention relates to a method for treating,preventing, inhibiting, reducing, eliminating, protecting or delayingthe onset of a disorder associated (either hereditary or acquired) withdysfunction of WASp in a subject in need thereof. More specifically, themethod may comprise administering to the subject a therapeuticallyeffective amount of at least one of the SMC modulators of WASp asdescribed by the invention or a pharmaceutically acceptable salt, estersor hydrate thereof or any analogs or derivatives thereof, anycombinations thereof, or of any vehicle, matrix, nano- or micro-particleor composition comprising the same.

In yet another aspect thereof, the invention provides the use of aneffective amount of at least one of the SMC modulators of WASp asdescribed by the invention or a pharmaceutically acceptable salt, estersor hydrate thereof, any analogs or derivatives thereof or anycombination thereof, or any vehicle, matrix, nano- or micro-particlecomprising the same, in the preparation of a composition for treating,preventing, inhibiting, reducing, eliminating, protecting or delayingthe onset of a hereditary or acquired disorders associated withdysfunction of WASp in a subject in need thereof.

The invention further provides SMC modulator of WASp degradation and/orstability. In more specific embodiments, the modulator of the inventionmay have the general formula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other selected to be absentor from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—, —(CH₂)—, —(CH₂)—O—,—NH—(CH₂)—, and each optionally substituted with C₁-C₅ alkyl, a ringsystem containing five to twelve atoms optionally substituted with C₁-C₅alkyl;

R₃ and R4 are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl.

A further aspect of the invention relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone of the SMC modulators of WASp as described by the invention or apharmaceutically acceptable salt, esters or hydrate thereof or anyanalogs or derivative thereof, or any vehicle, matrix, nano- ormicro-particle comprising the same, said composition optionally furthercomprises at least one pharmaceutically acceptable carrier/s,excipient/s, auxiliaries, and/or diluent/s.

Still further, the invention provides a kit comprising: a. at least oneSMC modulator as disclosed by the invention; and at least one of: b. atleast one chemotherapeutic agent; c. at least one biological therapyagent; and d. at least one agent that induces differentiation ofhematopoietic progenitor cells.

These and other aspects of the invention will become apparent by thehand of the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIG. 1A-1C. Structural model of WASp WH1 for virtual screening ofWASp-binding SMCs

FIG. 1A. shows an NMR model of the EVH1 domain of rat N-WASp conjugatedto a WIP polypeptide (PDB ID: 2IFS) that was virtually modified usingvirtual mutagenesis tool of Discovery Studio 3.0, allowing the model tobe representative of the WH1 domain of human WASp. The required site forSMCs binding is marked with a white circle.

FIG. 1B-1C. shows a ribbon and surface structures of WASp WH1 model.Lysine residues 76 and 81 are light gray and dark gray, respectively.

FIG. 2. Schematic workflow of virtual screening for WASp-binding SMCs Aschematic representation of the in silico prediction process. Rectanglesdenote data representative from a single protein-ligand interaction.Stacks of rectangles denote the data representative of all ligand modelsthat were used. Oval shapes denote the major programs that were used inthe process. The protein surface diagram denotes the modified NMR modelof the N-WASp WH1 domain in PDB format. Using the FAST algorithm, 3Dconformations of the various ligands were generated, and inputted toPatchDock with the modified NMR model. Features were extracted from theleading docking model and from the docking scores of all the modelsgenerated by PatchDock, and combined into a data vector. Finally, thepredictor classified each ligand-protein docking using the data vector,giving each a score denoting whether it is predicted to berepresentative of a possible binding.

FIG. 3A-3G. MST analysis validates the binding of virtually-screenedSMCs to WASp but not to its homologous, N-WASp and WAVE2 Diluted celllysates of YFP-WASp/N-WASp/WAVE2-expressing HEK 293T cells wereincubated with serially-diluted (100 μM-3 nM) SMCs. The mixed lysatesand SMCs were then loaded into standard-treated Monolith™ capillariesand the fluorescence of the samples was measured by the Monolith NT.115instrument at 60% MST power. Binding curves of WASp with SMC #6 (FIG.3A), SMC #30 (FIG. 3B), SMC #33 (FIG. 3C), SMC #34 (FIG. 3D) and Y-27632(FIG. 3E) or of N-WASp (FIG. 3F) or WAVE2 (FIG. 3G) with SMC #34 weregenerated by the NanoTemper Analysis 2.231 software. Normalizedfluorescence (hot fluorescence/initial fluorescence) is plotted as afunction of SMC concentration. Data shown are representative of at leastthree independent experiments.

FIG. 4A-4D. SMC of the invention upregulates WASp expression inplatelets and primary lymphocytes

FIG. 4A-4B. Freshly-isolated platelets were incubated with SMC#6 orcontrol at 37° C. for the indicated times. The platelets were eitherstimulated (+) with TRAP-6 (10 μM) or left unstimulated (−), followed bylysis. Lysates were analyzed for WASp expression by immunoblotting withanti-WASp and anti-GAPDH as loading control (FIG. 4A). Relative WASpexpression was determined by densitometric analysis and is presented ina summarizing graph of FIG. 4B. Error bars represent the SE from themean. P values are presented and were calculated by two-tailed Student'st-test. Data are representative of three independent experiments.

FIG. 4C-4D. Freshly-isolated PBMCs were incubated with SMC#6 or controlat 37° C. for the indicated times. The PBMCs were either co-stimulated(+) with anti-CD3 and anti-CD28 or left unstimulated (−), followed bylysis. Lysates were analyzed for WASp expression by immunoblotting withanti-WASp and anti-GAPDH as loading control (FIG. 4C). Relative WASpexpression was determined by densitometric analysis and is presented ina summarizing graph of FIG. 4D. Error bars represent the SE from themean. P values were calculated versus DMSO-treated control cells bytwo-tailed Student's t-test, and is indicated therein. Data arerepresentative of three independent experiments.

FIG. 5A-5D. WASp-binding SMCs upregulate WASp expression in T-cells,primary lymphocytes and platelets

Jurkat T-cells (FIG. 5A) or Freshly-isolated PBMCs (FIG. 5B) wereincubated for 15 min at 37° C. with either control or the indicatedWASp-binding SMCs, followed by co-stimulation with anti-CD3 andanti-CD28 and lysis. Freshly-isolated platelets were incubated for 15min at 37° C. with either control or the indicated WASp-binding SMCs,followed by stimulation with TRAP-6 (10 μM) and lysis (FIG. 5C). Lysateswere analyzed for WASp expression by immunoblotting with anti-WASp andanti-GAPDH as loading control. Western blot is representative of threeindependent experiments. FIG. 5D shows a summary of WASp expressionanalysis in PBMCs and platelets. Relative WASp expression was determinedby densitometric analysis. Error bars represent the SE from the mean.Data are representative of three independent experiments.

FIG. 6A-6D. WASp-binding SMCs upregulate WASp expression in platelets

FIG. 6A-6B. show freshly-isolated platelets that were incubated for 15min at 37° C. with either DMSO or the indicated WASp-binding SMCs,followed by stimulation (+) with TRAP-6 (10 μM) and lysis. Lysates wereanalyzed for WASp expression by immunoblotting with anti-WASp andanti-GAPDH as loading control (FIG. 6A).

Summarizing graph is presented in FIG. 6B.

FIG. 6C-6D. show the western blot of freshly-isolated plateletsincubated for 15 min at 37° C. with either DMSO or potent WASp-bindingSMCs that were stimulated with TRAP-6 (10 μM), followed by lysis (FIG.6C). Error bars represent the SE from the mean. Data are representativeof three independent experiments. Summarizing graph is presented in FIG.6D.

FIG. 7A-7G. Characterization of ubiquitylation-dependent degradationmechanism of WASp in platelets and megakaryocytes

Freshly-isolated platelets were stimulated with 10 μM of TRAP-6 for theindicated time points, followed by lysis. WIP (FIG. 7A) or WASp (FIG.7B) were immunoprecipitated from the lysates and analyzed by westernblot for co-immunoprecipitation, using anti-WASp, anti-WIP or anti-PKCθantibodies. WASp immunoprecipitates were immunoblotted for WASpubiquitylation using anti-ubiquitin (Ub) antibody (FIG. 7C).Ubiquitylated WASp appears as a smear of bands above the MW of 65 kDawith a prominent band at ˜81 kDa. FIG. 7D shows western blot analysis ofWASp expression in activated platelets, with (+) or without (−) theaddition of MG132 proteasome inhibitor. Blots are representative ofthree independent experiments. Meg-01 megakaryocyte cell line wasactivated in the presence (+) or absence (−) of MG132, followed by lysis(FIG. 7E). WASp ubiquitylation was determined by co-immunoprecipitationanalysis as in FIG. 7C. FIG. 7F illustrates Meg-01 cells that weretransfected with PKCθ-specific siRNA (+) or non-specific (NS) siRNA (−).24 h later, the cells were stimulated and lysed. PKCθ and WASp proteinexpression levels were analyzed by western blot with anti-PKCθ andanti-WASp antibodies, and with anti-GAPDH as loading control. Relativeprotein expression was determined by densitometric analysis and ispresented below the corresponding lanes. FIG. 7G. shows western blotanalysis of WASp expression in whole cell lysates (W.C.L) ofmegakaryocyte, with (+) or without (−) the addition of MG132 proteasomeinhibitor. Blots are representative of three independent experiments.

FIG. 8A-8D. WASp-binding SMCs up-regulate lymphocyte activation andproliferation Figures show 1×10⁶ PBMC cells/ml stimulated with PMA andlonomycin that were treated with DMSO or WASp-binding SMCs. Afterincubation, the cells were stained with PE-Cy5™-conjugated mouseanti-human CD69 and lymphocyte activation was measured by flow-cytometryas described in the Experimental procedures. Histograms of DMSO versusSMC #6 (FIG. 8A), SMC #30 (FIG. 8B), SMC #33(FIG. 8C), and SMC #34 (FIG.8D) are presented and represent three independent experiments.

FIG. 9A-9E. WASp-binding SMCs upregulate lymphocyte activation andproliferation

FIGS. 9A and 9B. show 1×10⁶ Jurkat T cells/ml, stimulated with PMA andlonomycin, were treated with WASp-binding SMC#6 or control. Afterincubation, the cells were stained with anti-activated LFA-1 antibody(KIM127), followed by staining with Alexa488-Fluor goat anti-mouse IgG₁secondary antibody (FIG. 9B). Cells were co-stained withPE-Cy5™-conjugated mouse anti-human CD69 and lymphocyte activation wasmeasured by flow-cytometry as described in the Experimental procedures(FIG. 9A). Histograms of negative control versus SMC#6 are presented.

FIG. 9C. shows 1×10⁶ PBMC cells/ml, stimulated with PMA and lonomycinthat were treated with WASp-binding SMC#6 or DMSO as negative control.After incubation, the cells were stained with anti-activated LFA-1antibody (KIM127), followed by staining with Alexa488-Fluor goatanti-mouse IgG₁ secondary antibody, and lymphocyte activation wasmeasured by flow-cytometry as described in the Experimental procedures.Histogram of negative control versus SMC#6 is presented.

FIG. 9D. shows the mean relative proliferation of stimulated JurkatT-cells, treated with the indicated WASp-binding SMCs or DMSO asnegative control from three independent experiments. Error barsrepresent the SE from the mean.

FIG. 9E. shows the mean relative proliferation of stimulated PBMCs,treated with the indicated WASp-binding SMCs or DMSO as negative controlfrom three independent experiments. Error bars represent the SE from themean.

FIG. 10A-10C. WASp-binding SMCs enhance the migration of lymphocytes onICAM-1

Jurkat T cells were pretreated with DMSO or the indicated WASp-bindingSMCs, and cell migration on ICAM-1/SDF-1α-coated cover glasses wastracked over a 20-min period, as described in Experimental procedures.Each line in FIG. 10A represents one cell. The tracks of fiverepresentative cells from each treatment are presented.

FIG. 10B. shows mean cell displacement (DMSO: 17.25±1.58 n=337; SMC #6:29.52±2.96 n=214; SMC #30: 41.07±4.69 n=55; SMC #33: 32.92±4.28 n=50;SMC #34: 48.91±6 n=60). Error bars represent the SE from the mean. Pvalues versus DMSO-treated control cells are presented and werecalculated by two-tailed Student's t-test.

FIG. 10C. illustrates the final frame of cells treated with DMSO or withthe indicated WASp-binding SMCs.

FIG. 11A-11D. WASp-binding SMCs increase intra-cellular calciumconcentration in platelets Freshly-isolated human platelets wereincubated with either control DMSO or the indicated WASp-binding SMCs,specifically, SMC#6 (FIG. 11A), SMC#30 (FIG. 11B), SMC#33 (FIG. 11C) andSMC#34 (FIG. 11D). Platelets were stimulated with TRAP-6 and calciumlevels were measured by spectrofluorometer, as described in Experimentalprocedures. Data are representative of three independent experiments.

FIG. 12A-12D. WASp-binding SMCs decrease WASp ubiquitylation inplatelets and primary lymphocytes FIGS. 12A and 12B. showfreshly-isolated platelets that were either stimulated (+) with TRAP-6(10 μM) or unstimulated (−) and pre-incubated at 37° C. for 15 min witheither DMSO or the indicated WASp-binding SMCs (FIG. 12A—SMC #6; FIG.12B—SMC #33), followed by lysis. Lysates were subjected toimmunoprecipitation with anti-WASp antibody and were analyzed by westernblotting with anti-ubiquitin (Ub) and with anti-WASp as precipitationcontrol. Ubiquitylated WASp appears as a smear of bands above themolecular weight (MW) of 65 kDa with a prominent band at ˜81 kDa. Blotsare representative of three independent experiments.

FIGS. 12C and 12D. show freshly-isolated PBMCs that were eitherco-stimulated (+) with anti-CD3 and anti-CD28 or unstimulated (−). ThePBMCs were pre-incubated at 37° C. for 15 min with either DMSO or theindicated WASp-binding SMCs (FIG. 12C—SMC #6; FIG. 12D—SMC #33),followed by lysis. WASp ubiquitylation was determined byimmunoprecipitation as in FIG. 12A-12B. Ubiquitylated WASp appears as asmear of bands above the MW of 65 kDa with a prominent band at ˜81 kDa.Blots are representative of three independent experiments.

FIG. 13A-13B. FRET analysis of the WIP-WASp interaction in activatedT-cells treated with WASp-binding SMC#6, SMC#30, SMC#33 or SMC#34 FIG.13A: Jurkat T cells stably expressing CFP-WASp and YFP-WIP were treatedwith WASp-binding SMC#6, SMC#30, SMC#33, SMC#34 or control. The cellswere then plated on stimulatory coverslips (coated with an anti-CD3antibody), and fixed after 5 min. Cells were imaged by confocalmicroscope, and FRET efficiency was measured by the donor-sensitizedacceptor emission technology (see Experimental procedures for details).

FIG. 13B: graph summarizing the percentage FRET efficiency in cellsplated on the stimulatory coverslips, with the indicated treatment. Dataare from four independent experiments. Error bars represent the SE fromthe mean. P values were calculated by two-tailed Student's t-test.

FIG. 14A-14E. WASp-binding SMCs restore WASp expression of commonWAS/XLT WASp mutations

FIG. 14A. presents a FACS analysis of Jurkat T-cells expressing YFP-WASpmutants. FIG. 14B. shows Jurkat T-cells stably expressing YFP-WASpmutants that were pretreated with either negative control or theindicated WASp-binding SMCs. The cells were then co-stimulated withanti-CD3 and anti-CD28, followed by lysis. Lysates were analyzed forWASp expression by immunoblotting with anti-WASp and anti-GAPDH asloading control. Relative protein expression was determined bydensitometric analysis and is presented below the corresponding lanes.Western blots are representative of three independent experiments.

FIG. 14C. shows western blot analysis of Jurkat T-cell line which haveno expression of endogenous WASp wt (WAS^(−/−)). Western blot isrepresentative of three independent experiments.

FIG. 14D. shows WASp knockout Jurkat T-cell lines that express onlyexogenous YFP-WASp harboring the common human WAS and XLT mutations,R86C (WAS^(−/−)/WASp R86C) or Y107C (WAS^(−/−)/WASp Y107C) that werepretreated with either negative control or SMC#34. The cells were thenco-stimulated with anti-CD3 and anti-CD28 and analyzed by western blotas described in FIG. 14B. Endogenous WASp displays a band at 65 kDa andexogenous YFP-WASp displays a band at 92 kDa. Relative proteinexpression was determined by densitometric analysis and is presentedbelow the corresponding lanes.

FIG. 14E. shows WASp-binding SMC up-regulates lymphocyte activation.1×10⁶ WASp knockout Jurkat T-cell line that expresses only exogenousYFP-WASp harboring the common human WAS and XLT mutation, R86C(WAS−/−/WASp R86C) was pretreated with either negative control orSMC#34. Cells that completely lack WASp expression (WAS^(−/−)) were alsotreated with the control. The cells were then stimulated with PMA andlonomycin. After incubation, the cells were stained withPE-Cy5™-conjugated mouse anti-human CD69 and lymphocyte activation wasmeasured by flow cytometry as described in Experimental procedures.Histogram of negative control versus SMC#34 is presented.

FIG. 15A-15C. WASp-binding SMC#34 enhances the migration of lymphocytesstably expressing common human WAS/XLT WASp mutations

WASp knockout Jurkat T-cell lines that express only exogenous YFP-WASpR86C (WAS^(−/−)/WASp R86C) or Y107C (WAS^(−/−)/WASp Y107C) werepretreated with SMC#34 in comparison to cells treated with the control.Cells that completely lack WASp expression (WAS^(−/−)) were also treatedwith the control. Cellular migration was tracked over a 20-min period onICAM-1/SDF-1α-coated cover glasses, as described in Experimentalprocedures.

FIG. 15A. The last frame in the movie of the control or the indicatedWASp-binding SMCs.

FIG. 15B. Each line represents one cell. The tracks of fiverepresentative cells from each treatment are presented.

FIG. 15C. Mean cell displacement (WAS^(−/−)/WASp R86C, DMSO negativecontrol: 17.74±2.46 n=110, SMC#34: 53.65±4.88 n=138; WAS^(−/−)/WASpY107C, DMSO negative control: 17.75±2.06 n=138, SMC#34: 74.24±8.16n=100; WAS^(−/−), DMSO: 5.15±1.09 n=88).

Error bars represent the SE from the mean. P values of SMC-treated cellsversus DMSO-treated control cells are presented and were calculated bytwo-tailed Student's t-test.

FIG. 16. Proposed model for the mechanism of action by whichWASp-binding SMCs protect WASp from degradation

WASp degradation sites, lysine residues 76 and 81 are located in apocket at the N′-terminus of WASp. In normal naïve cells WIP is tightlybound to WASp and protects it from ubiquitylation and degradation (upperscheme). WIP protection is relieved following cellular activation oralternatively due to WAS/XLT mutations, which abrogate the WIP-WASpinteraction. Unprotected, lysine residues 76 and 81 are ubiquitylatedand WASp is degraded (lower right). WASp-binding SMCs mask WASpdegradation sites by binding in the close proximity of the pocket at theN′-terminus. This masking protects WASp from ubiquitylation anddegradation (lower left).

DETAILED DESCRIPTION OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

As discussed herein before, so far, the attempts for curative treatmentsfor WAS/XLT patients were focused on restoring WASp expression andfunction in hematopoietic cells, either by hematopoietic stem celltransplantation (HSCT) or gene therapy. Despite being the only approvedrestorative treatment for WAS, HSCT is a customized treatment, whichrequires the availability of a matching donor and early intervention.The graft source (bone marrow, cord blood or peripheral blood) and theintensity of preparatory conditioning are also complex considerationthat must be taken prior to transplantation. Post-transplantcomplications may include autoimmunity, malignancies, viral infections,graft-versus-host disease (GVHD) and infertility. Gene therapy, which isin fact an autologous gene-modified HSCT, is an emerging treatmentintended to bypass the obstacle of finding a matching donor, and toavoid GVHD. Ex vivo gene correction in this approach is facilitated byvarious viral vectors that integrate the correct gene to patient-derivedhematopoietic progenitor cells, which are then reinfused back to thepatient. Unfortunately however, previous clinical studies reported theonset of leukemia in most of the patients who undergone gene therapywith γ-retroviral vectors, due to vector-related insertional oncogenesis(Aiuti et al, 2013). Current efforts at improving safety and efficacy ofgene therapy are focusing on lentiviral vectors, which show someclinical improvement with no insertional oncogenesis-associated eventsrecorded to date (Hacein-Bey Abina et al, 2015). However, more time isneeded to follow-up patients in order to assess long-term safety andefficacy. Still, gene therapy carries the other risks of allogeneicHSCT, such as infections and infertility due to the preparatoryconditioning.

These intensive treatments are considered excessively risky for XLTpatients, because XLT patients have good long-term survival, with lifeexpectancy that is not substantially different from the normalpopulation. However, XLT patients suffer from severe disease-relatedcomplications, with only 27% of XLT patients reach the age of 60 withoutexperiencing severe disease-related events (Albert et al, 2010). Thus,there is currently no generally accepted treatment policy for XLTpatients. Splenectomy is offered to WAS/XLT patients in order toincrease platelet count, and, therefore to reduce bleeding events.However, splenectomy is associated with increased risk of infections,requiring the patients to receive life-long antibiotic prophylaxis.Moreover, WAS/XLT patients who underwent splenectomy are not compatiblefor further HCST because they have an increased morbidity and mortalitypost-HSCT, due to viral infections (Buchbinder et al, 2014).

The inventors report herein that small molecule compounds (SMCs) restoreWiskott Aldrich Syndrome protein (WASp) expression and function. Theinventors further revealed the mechanism underlying this effect of thenovel SMCs described herein. More specifically, in normal resting cellsWASp is auto inhibited by its tight bond to a protective protein—WIP,which masks WASp degradation sites at lysine residues 76 and 81. Incontrast, WAS gene mutations trigger a conformational change thatreleases WIP protection from the degradation pocket of WASp,subsequently resulting in WASp degradation by uncontrolledubiquitylation. The resulting WASp deficiency affects a broad spectrumof cell functions, and the severity of the disease is determined by thelevel of WASp expression. The inventors used an in-silico predictor toscreen potential WASp-binding SMCs that protect it from degradation. Thebinding of potent SMCs was validated using microscale thermophoresis(MST) technology.

More specifically, in the present disclosure, the inventors providenovel WASp-binding SMCs (EXAMPLE 1, Table 1, FIG. 1, FIG. 3) thatpotentially protect WASp from degradation, via blocking theubiquitylation of lysine residues 76 and 81 located in a pocket at WASpWH1 domain. Moreover, the inventors show that by reducing WASpubiquitylation, the selected SMCs up-regulate WASp expression in T-celllines, primary lymphocytes and platelets (FIG. 5, FIG. 12). Thisupregulation of WASp expression by WASp-protecting SMCs, enhancedWASp-dependent cellular function, including cellular activation,intra-cellular calcium influx, proliferation and migration (FIG. 8—FIG.11). Strikingly, these SMCs restored the function of mutantWASp-expressing cells (FIG. 14—FIG. 15). Therefore, the inventors haverevealed a novel function of the selected SMCs, namely, attenuation ofWASp degradation by binding to WASp. The findings of the invention arehighly valuable and may lead to the development of new therapeuticstrategies which target WASp expression. Moreover, these findings form apromising treatment modality for hereditary WASp dysfunction disorderssuch as WAS/XLT as well as for acquired WASp dysfunction disorders aswill be elaborated below.

Thus, a first aspect of the invention relates to an effective amount ofat least one small molecule compound (SMC) modulator or any vehicle,matrix, nano- or micro-particle comprising the same, for use in a methodfor modulating the degradation of Wiskott-Aldrich Syndrome protein(WASp) in a cell. In more specific embodiments, the SMC of the inventionmay have the general formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, branched C₁-C₂ alkyl, C₂-C₁₂ alkenyl,C₂-C₁₂alkynyl, straight C₁-C₂ alkoxy, branched C₁-C₁₂ alkoxy, a ringsystem containing five to twelve atoms, each optionally substituted byat least one of straight C₁-C₅ alkyl, branched C₁-C₅ alkyl, halide,hydroxyl, ester, ether, amide, amine, nitro, CF₃,—C(═O)—O—(CH₂)_(n)—CH₃, R₅, or —NH—C(═O)—R₅, R₅ is an a ring systemcontaining five to twelve atoms optionally substituted by at least onehalide or straight C₁-C₅ alkyl or branched C₁-C₅ alkyl;

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to twelve membered saturated or unsaturated ring that mayoptionally include N, O, S, NH, C═N, C═O, S═O or SO₂ and may beoptionally be substituted with at least one of straight or branchedC₁-C₅ alkyl, hydroxyl, halide and cyano;

L1 and L2 are each independently from each other selected to be absentor from —(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—,—(CH₂)_(n)—O—, S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n),—(CH₂)_(n)—N—C(═O)—, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—, —NH—(CH₂)_(n)—,—C(═O)—NH—(CH₂)_(n)—; —S—S—(CH₂)_(n)—; —O—(CH₂)_(n)—; —NH—(CH₂)_(n)—;C(═O)—(CH₂)_(n)—; —S—(CH₂)_(n)—; —NH—S(═O)_(n)—(CH₂)_(n)—,—CH₂—S—C(O)—NH—CH₂—, —(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—(NH)_(n)—C(═O)—, —(CH₂)_(n)—N—C(═O)-L1 andL2 may be each independently from each other optionally substituted withC₁-C₅ alkyl, a ring system containing five to twelve atoms optionallysubstituted with C₁-C₅ alkyl

each n, is an integer being independently from each other selected frombe 0 to 5;

R₃ and R4 are each independently from each other be absent or selectedfrom a ring system containing five to 15 atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), (O)₂, —C(O)—CH₃, —C(O)—O—CH₃, halide, CF₃, nitro, amide, or R₅, R₅is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl or branchedC₁-C₅ alkyl.

In some embodiments, a ring system containing five to twelve atoms orfive to 15 atoms may be substituted with one or more substituents, incertain embodiments one, two, three or four substituents as detailedherein. It should be noted that when referring to ring system five totwelve atoms or five to 15 atoms, it may optionally include least one ofN, O, S, NH, C═N, C═O, S═O, or SO₂.

In accordance with this aspect, in some specific embodiments, thecompound of general Formula I may provide a compound having the generalformula (I′):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, branched C₁-C₁₂ alkyl, a ring system containingfive to twelve atoms, each optionally substituted by at least one ofhalide, amide, amine, nitro, —C(═O)—O—(CH₂)_(n)—CH₃; R₅ or —NH—C(═O)—R₅,R₅ is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl or branchedC₁-C₅ alkyl;

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to twelve membered saturated or unsaturated ring that mayoptionally include at least one of N, NH and may be optionally besubstituted with at least one of straight or branched C₁-C₅ alkyl,

L1 and L2 are each independently from each other selected from—(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—, —(CH₂)_(n)—O—,S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—; —(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—, —NH—(CH₂)_(n)—, L1 and L2 may beeach independently from each other optionally substituted with C₁-C₅alkyl, a ring system containing five to twelve atoms optionallysubstituted with C₁-C₅ alkyl, each n, is an integer being independentlyfrom each other selected from be 0 to 5;

R₃ and R4 are each independently from each other absent or selected froma ring system containing five to 15 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S), (O)₂,or R₅, R₅ is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight or branched C₁-C₅ alkyl.

In accordance with some embodiments, at least one of R₁ and R₂ isindependently from each other selected from H, a ring system containingfive to twelve atoms. In accordance with some other embodiments, atleast one of R₁ and R₂ is independently from each other selected from H.In some other embodiments, at least one of R₁ and R₂ is independentlyfrom each other selected from a ring system containing five to twelveatoms, which is optionally substituted with halide, amide, nitro,—C(═O)—O—(CH₂)_(n)—CH₃, R₅, or —NH—C(═O)—R₅. In some embodiments, R₅ isan aryl or thiophene, optionally substituted with at least one halide.

In some other embodiments, the ring system of at least one of R₁ and R₂may be an aryl (aromatic ring) or aliphatic ring (non-aromatic ring). Insome further embodiments, at least one of R₁ and R₂ may be C₅-C₁₂saturated cycloalkyl, C₅-C₁₂ saturated cycloalkylene, C₅-C₁₂ aryl orC₅-C₁₂ arylene. In some further embodiments, the ring system of at leastone of R₁ and R₂ may contain at least two carbon atoms and may includeat least one heteroatom ring. In some further embodiments, at least oneof R₁ and R₂ may be heteroaryl, heteroarylene, heterocycloalkylene orheterocycloalkyl. In some further embodiments, at least one of R₁ and R₂may be C₂-C₁₂ heterocycloalkyl ring, C₂-C₁₂ heteroaryl or C₂-C₁₂heteroarylene. In some embodiments, the heteroatom may be N, O, S. Insome other embodiments, at least one of R₁ and R₂ may be thiazole,cyclopentane, naphthalene, aryl, each optionally substituted with—C(═O)—O—(CH₂)—CH₃, halide, nitro or —NH—C(═O)—R₅. In some embodiments,R₅ is an aryl substituted with at least one halide.

In some other embodiments, at least one of R₁ and R₂ may be thiazole. Insome other embodiments, at least one of R₁ and R₂ may be cyclopentane.In some other embodiments, at least one of R₁ and R₂ may be naphthalene.In some other embodiments, at least one of R₁ and R₂ may be an arylsubstituted with —C(═O)—O—(CH₂)—CH₃. In some other embodiments, at leastone of R₁ and R₂ may be an aryl substituted with at least one halide,preferably F. In some other embodiments, at least one of R₁ and R₂ maybe an aryl substituted with at two F atoms. In some other embodiments,at least one of R₁ and R₂ may be an aryl substituted with one F and oneC₁ groups.

In some other embodiments, at least one of R₁ and R₂ may be an arylsubstituted with nitro.

In some other embodiments, at least one of R₁ and R₂ may be—NH—C(═O)—R₅. In some embodiments, R₅ is an aryl substituted with atleast one halide. In some further embodiments, at least one of R₁ and R₂may be benzoic acid methyl ester.

In some other embodiments, R₁ and R₂ are each independently from eachother selected from straight or branched C₁-C₁₂ alkyl optionallysubstituted by at least one of amide or thiophene In some otherembodiments, at least one of R₁ or R₂ may be CH₂—CH substituted by atleast one of dimethyl amine or thiophene.

In some other embodiments, R₁ and R₂ are each independently from eachother selected from straight C₁-C₁₂ alkyl. In some embodiments, R₁ andR₂ are each independently from each other selected from CH₃.

In accordance with some embodiments, R₁ and R₂ together with thenitrogen atom they are connected to form a five or six memberedsaturated or unsaturated ring that may optionally include at least oneof N and may be optionally be substituted with at least one of straightor branched C₁-C₅ alkyl. In some embodiments, R₁ and R₂ together withthe nitrogen atom they are connected to form piperidine or piperazine,each optionally substituted with at least one straight or branched C₁-C₅alkyl. In some embodiments, R₁ and R₂ together with the nitrogen atomthey are connected to form piperidine or piperazine substituted with atleast one CH₃ group, at times two CH₃ groups, at times three CH₃ groups.

In some embodiments, R₁ and R₂ together with the nitrogen atom they areconnected to form piperazine substituted with two CH₃ groups. In someembodiments, R₁ and R₂ together with the nitrogen atom they areconnected to form piperazine substituted with one CH₃ group.

In some embodiments, L1 may be absent or may be selected from—CH₂—CH₂—C(O)—N—CH₂—C(O)—NH—CH₂—, —(CH₂)—S—, —CH₂—, —(CH₂)—N—C(═O)—,—(CH₂)—S—, —(CH₂)—S—(CH₂)_(n)—C(O)—NH—(CH₂)—, —(CH₂)—NH—NH—C(═O)—,—NH—(CH₂)—, each optionally substituted with ethyl, methyl, an aryloptionally substituted with a methyl or ethyl.

In some embodiments, L2 may be absent or may be selected from —(CH₂)—O—,S(O)₂, S(O)₂—N—, CH₂, —S(O)₂—NH—(CH₂)—, NH each optionally substitutedwith a methyl, an ethyl, an aryl optionally substituted with a methyl,ethyl.

In some other embodiments, each of R₃ and R₄ may be absent. In someother embodiments, each of R₃ and R₄ may be selected from a ring systemcontaining five to 15 atoms, optionally substituted as described herein.In some further embodiments, each of R₃ and R₄ may be selected from aring system containing five to twelve atoms, optionally substituted asdescribed herein.

In some other embodiments, the ring system of at least one of R₃ and R₄may be an aryl (aromatic ring) or aliphatic ring (non-aromatic ring). Insome further embodiments, at least one of R₃ and R₄ may be C₅-C₁₂saturated cycloalkyl, C₅-C₁₂ saturated cycloalkylene, C₅-C₁₂ aryl orC₅-C₁₂ arylene. In some further embodiments, the ring system of at leastone of R₃ and R₄ may contain at least two carbon atoms and may includeat least one heteroatom ring. In some further embodiments, at least oneof R₃ and R₄ may be heteroaryl, heteroarylene, heterocycloalkylene orheterocycloalkyl. In some further embodiments, at least one of R₃ and R₄may be C₂-C₁₂ heterocycloalkyl ring, C₂-C₁₂ heteroaryl or C₂-C₁₂heteroarylene. In some embodiments, the heteroatom may be N, O, S.

In some other embodiments, at least one of R₃ and R₄ may be thiazole,cyclopentane, naphthalene, triazine, triazole, piperidine, piperazine,quinoline, isoquinoline, phthalazine, tetrahydro-quinoline,tetrahydro-quinazoline pyridine, oxadiazole, aryl, each optionallysubstituted with at least one of C₁-C₅ alkyl, (═O), (═S), —C(O)—CH₃,—C(O)—O—CH₃, halide, nitro, aryl, pyridine, morpholine, S(O)₂-amide.

In some other embodiments, at least one of R₃ and R₄ may be triazole,triazine, piperidine, piperazine, quinoline, isoquinoline, phthalazine,pyridine, tetrahydro-quinoline, tetrahydro-quinazoline oxadiazole, aryl,each optionally substituted with at least one of C₁-C₅ alkyl, (═O),(═S), —C(O)—CH₃, —C(O)—O—CH₃, pyridine, morpholine, S(O)₂-amide.

In some other embodiments, R₃ may be triazole, triazine, piperidine,phthalazine, oxadiazole, aryl, each optionally substituted with at leastone of CH₃, (═O), (═S), morpholine.

In some other embodiments, R₄ may be piperidine, piperazine, quinoline,isoquinoline, phthalazine, tetrahydro-quinoline, pyridine,tetrahydro-quinazoline aryl, each optionally substituted with at leastone of C₁-C₅ alkyl, (═O), (═S), —C(O)—CH₃, —C(O)—O—CH₃, morpholine,S(O)₂-amide.

In some embodiments, L1 is absent and R₃ is an aryl, optionallysubstituted.

In accordance with such embodiments, the present disclosure provides thecompound of Formula I that may in some embodiments be presented as acompound having the general formula (II):

wherein R1, R2, L2 and R4 are as defined above for a compound having thegeneral formula (I) or for a compound having the general formula (I′).

In some embodiments, L2 is selected from C(O) and S(O)₂. In someembodiments, L1 is absent, R₃ is an aryl and L2 is selected from C(O)and S(O)₂.

In accordance with such embodiments, the present disclosure provides acompound having the general formula (III), that is derived from thecompound of Formula I:

wherein R1, R2 and R4 are as defined above for a compound having thegeneral formula (I) or for a compound having the general formula (I′).

Still further aspects of the invention relate to an effective amount ofat least one small molecule compound (SMC) modulator or any vehicle,matrix, nano- or micro-particle comprising the same, for use in a methodfor modulating the degradation of Wiskott-Aldrich Syndrome protein(WASp) in a cell. In more specific embodiments, the modulator of theinvention may have the general formula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other may be absent orselected from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—, —(CH₂)—, —(CH₂)—O—,—NH—(CH₂)—, and each optionally substituted with C₁-C₅ alkyl, a ringsystem containing five to twelve atoms optionally substituted with C₁-C₅alkyl;

R₃ and R₄ are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl.

In some embodiments, in the SMC modulator for use in accordance with theinvention, the R₁ and R₂ are each independently from each other selectedfrom H, C₅-C₇ saturated cycloalkyl, C₅-C₇ saturated cycloalkylene, C₅-C₇aryl or C₅-C₇ arylene, each optionally substituted by at least one aring system containing five to seven atoms optionally substituted by atleast one halide or straight C₁-C₅ alkyl,

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive or six membered saturated or unsaturated ring optionally include atleast one of N and optionally substituted with at least one of straightC₃-C₅ alkyl,

L1 may be either absent or may be selected from —CH₂—S—,—CH₂—CH₂—C(O)—N—CH₂—C(O)—NH—CH₂, —CH₂—, each optionally substituted withethyl, methyl, an aryl optionally substituted with a methyl or ethyl, L2is absent or —CH₂—O—, CH₂;

R₃ and R₄ are each independently from each other absent or selected fromC₅-C₁₂ heterocycloalkyl ring, C₂-C₁₅ heteroaryl or C₂-C₁₅heteroarylene.In some embodiments, the heteroatom may be N, O, S, each optionallysubstituted with at least one of straight C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl.

In some further embodiments, in the SMC modulator for use in accordancewith the invention, the R₁ and R₂ may be each independently from eachother selected from H, cyclopentane, or thiazole. Alternatively, R₁ andR₂ together with the nitrogen atom they are connected to form a sixmembered unsaturated ring substituted with at least one methyl,

L1 is absent or is selected from —CH₂—S—,—CH₂—CH₂—C(O)—N—CH₂—C(O)—NH—CH₂—, substituted with an aryl optionallysubstituted with a methyl

L2 is absent, CH₂, or —CH₂—O—,

R₃ and R₄ are each independently from each other absent or selected fromthe group consisting of piperidine, piperazine, quinoline, isoquinoline,phthalazine, tetrahydro-quinoline, pyridine, tetrahydro-quinazoline 1,2, 4-triazole, oxadiazole quinoline, pyridine, phenyl, naphthalenesubstituted with —C(O)—CH₃ or methyl.

In some particular embodiments of the SMC modulator for use inaccordance with the invention, the compound of Formula XI, may provide acompound having the general formula (XII):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl;

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered unsaturated ring optionally include at least oneof N, O and optionally substituted with at least one of straight C₁-C₅alkyl,

L1 and L2 each independently from each other may be absent or selectedfrom —(CH₂)_(n)—, (CH₂)_(n)—S—, —(CH₂)_(n)—O—, and may be optionallysubstituted with C₁-C₅ alkyl, n may be an integer selected from 0 or 1,

R₃ is a ring system containing five atoms, optionally substituted withat least one straight C₁-C₅ alkyl, or R₅, R₅ is an a ring systemcontaining five to seven atoms optionally substituted by at least onehalide or straight C₁-C₅ alkyl,

R₄ is absent or selected from a ring system containing five to 12 atoms,each optionally substituted with at least one of straight C₁-C₅ alkyl.

In accordance with some embodiments, of the SMC modulator for use, R₁and R₂ are each independently from each other selected from H, straightC₂₋₄ alkyl, cycloalkyl, each optionally substituted by at least one aring system containing five to seven atoms, or

R₁ and R₂ together with the nitrogen atom they are connected to form afive or six membered unsaturated ring optionally be substituted with atleast one of methyl,

L1 is —CH₂—S—, L2 is absent or —CH₂—O—,

R3 is a 5-membered aromatic ring, selected from the group consisting ofoxazole, 1, 2, 4-traizole, oxadiazole, each optionally substituted by atleast one methyl,

R4 is absent or selected from the group consisting of quinoline,pyridine, phenyl, naphthalene.

In yet some further specific embodiments, the SMC modulator for use inaccordance with the invention wherein R₁ and R₂ together with thenitrogen atom they are connected to form a six membered unsaturated ringsubstituted with at least one methyl,

L1 is —CH₂—S—, L2 is absent or —CH₂—O—, R₃ is 1, 2, 4-triazole,oxadiazole, each optionally substituted by at least one methyl, R₄ isabsent or selected from the group consisting of quinoline, pyridine.

In some particular embodiments, specific and non-limiting examples ofthe SMC modulators for use of the invention, or pharmaceuticallyacceptable salts or hydrates of the compounds of Formula XI, or in somefurther specific embodiments, pharmaceutically acceptable salts orhydrates of the compounds of Formula XII, may include:

1-(2,6-Dimethyl-piperidin-1-yl)-2-[5-(quinolin-8-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-ethanone(designated herein as SMC 34).

In yet another alternative, the SMC modulators for use of the inventionmay be

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-3-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(designated herein as SMC 34.7).

In yet some further particular embodiments, specific and non-limitingexamples of the SMC modulators for use of the invention, orpharmaceutically acceptable salts or hydrates of the compounds ofFormula XI, or in some further specific embodiments, pharmaceuticallyacceptable salts or hydrates of the compounds of Formula XII, mayinclude:

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-m-tolyloxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.1)

2-(5-Methyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-piperidin-1-yl-ethanone(SMC 34.3);

N-Cyclohexyl-2-[5-(naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetamide(SMC 34.4);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.5);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-pyridin-3-yl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.6);

2-(5-Phenoxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-pyrrolidin-1-yl-ethanone(SMC 34.8);

2-(Quinolin-8-yloxymethyl)-oxazole-4-carboxylic acid(tetrahydro-pyran-2-ylmethyl)-amide (SMC 34.10)

2-[4-Phenyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-1-piperidin-1-yl-ethanone(SMC 34.11)

1-Piperidin-1-yl-2-(5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.12);

2-[4-Methyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide(SMC 34.13).

In yet some further particular embodiments, a further example of a SMCmodulator that may be used by the invention, may be the compound

[5-(Naphthalen-1-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetic acidmethyl ester (SMC 34.9).

In yet some further particular embodiments, a further example of a SMCmodulator that may be used by the invention, may be the compound

8-(5-Isopropyl-[1,3,4]oxadiazol-2-ylmethoxy)-quinoline (34.2)

In yet some further particular embodiments, the SMC modulator used bythe invention may be any compound defined by Formula XI, Formula XII,with the proviso that the compound is not any of the compounds detailedbelow:

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-m-tolyloxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.1)

2-(5-Methyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-piperidin-1-yl-ethanone(34.3);

N-Cyclohexyl-2-[5-(naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetamide(34.4);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(34.5);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-pyridin-3-yl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(34.6);

2-(5-Phenoxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-pyrrolidin-1-yl-ethanone(34.8);

2-(Quinolin-8-yloxymethyl)-oxazole-4-carboxylic acid(tetrahydro-pyran-2-ylmethyl)-amide (SMC 34.10);

2-[4-Phenyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-1-piperidin-1-yl-ethanone(SMC 34.11);

1-Piperidin-1-yl-2-(5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(34.12);

2-[4-Methyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide(34.13);

In yet some further particular embodiments, the SMC modulator that maybe used by the invention, may be any of the compounds disclosed by theinvention provided that said compound is not the compound:

[5-(Naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetic acidmethyl ester (34.9).

In yet some further particular embodiments, the SMC modulator that maybe used by the invention, may be any of the compounds disclosed by theinvention provided that said compound is not the compound:

8-(5-Isopropyl-[1,3,4]oxadiazol-2-ylmethoxy)-quinoline (34.2);

In yet some alternative particular embodiments of the SMC modulator foruse in accordance with the invention, the compound of Formula XI, mayprovide a compound having the general formula (XIV):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H andstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other may be absent orselected from —(CH₂)—S—, —(CH₂)—, —(CH₂)—O—,

R₃ and R₄ are each independently from each other absent or selected froman aryl or heteroaryl group optionally substituted with at least one ofstraight or branched C₁-C₅ alkyl, halide, nitro and cyano.

In some embodiments, in the SMC modulator for use in accordance with theinvention, R₁ and R₂ are each independently from each other selectedfrom H, methyl and ethyl, at times R₁ and R₂ are each independently fromeach other selected from H and methyl, L1 may be —CH₂—S—, L2 may be—CH₂—O—; R₃ and R₄ are each independently from each other absent orselected from an aryl or heteroaryl group optionally substituted with atleast one of straight or branched C₁-C₅ alkyl, halide, nitro and cyano.

In some further embodiments, in the SMC modulator for use in accordancewith the invention, the R₁ and R₂ may be each independently from eachother selected from H and methyl, L1 is —CH₂—S—, L2 is, —CH₂—O—, R₃ isselected from the group consisting of thiazole, [1,3,4]thiadiazole,[1,3,4]oxadiazole, and R₄ is phenyl.

In yet some alternative particular embodiments of the SMC modulator foruse in accordance with the invention, the compound of Formula I, mayprovide a compound having the general formula (XV):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl and branched C₁-C₁₂ alkyl;

L1 and L2 are each independently from each other selected to be absentor from —(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—,—(CH₂)_(n)—O—, S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n),—(CH₂)_(n)—N—C(═O)—, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—, —NH—(CH₂)_(n)—,—C(═O)—NH—(CH₂)_(n)—; —S—S—(CH₂)_(n)—; —O—(CH₂)_(n)—; —NH—(CH₂)_(n)—;C(═O)—(CH₂)_(n)—; —S—(CH₂)_(n)—; —NH—S(═O)_(n)—(CH₂)_(n)—,—CH₂—S—C(O)—NH—CH₂—, —(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—(NH)_(n)—C(═O)—, —(CH₂)_(n)—N—C(═O)— L1and L2 may be each independently from each other optionally substitutedwith C₁-C₅ alkyl, a ring system containing five to twelve atomsoptionally substituted with C₁-C₅ alkyl

each n, is an integer being independently from each other selected frombe 0 to 5;

R₃ and R₄ are each independently from each other be absent or selectedfrom a ring system containing five to 15 atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), (O)₂, —C(O)—CH₃, —C(O)—O—CH₃, halide, CF₃, nitro, amide, or R₅, R₅is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl or branchedC₁-C₅ alkyl.

In some embodiments, R₁ and R₂ are each independently from each otherselected from H or straight C₁-C₅ alkyl, L1 and L2 are eachindependently from each other selected to be absent or from—(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—, —(CH₂)_(n)—O—,S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n), —(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)—, —NH—(CH₂)_(n)—, —C(═O)—NH—(CH₂)_(n)—;—S—S—(CH₂)_(n)—; —O—(CH₂)_(n)—; —NH—(CH₂)_(n)—; C(═O)—(CH₂)_(n)—;—S—(CH₂)_(n)—; —NH—S(═O)_(n)—(CH₂)_(n)—, —CH₂—S—C(O)—NH—CH₂—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—N—C(═O)—, S(O)₂—N—(CH₂)_(n),—(CH₂)_(n)—(NH)_(n)—C(═O)—, —(CH₂)_(n)—N—C(═O)— L1 and L2 may be eachindependently from each other optionally substituted with C₁-C₅ alkyl, aring system containing five to twelve atoms optionally substituted withC₁-C₅ alkyl; each n, is an integer being independently from each otherselected from be 0 to 5;

R₃ and R₄ are each independently from each other be absent or selectedfrom a ring system containing five to 15 atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), (O)₂, —C(O)—CH₃, —C(O)—O—CH₃, halide, CF₃, nitro, amide, or R₅, R₅is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl or branchedC₁-C₅ alkyl.

In some other embodiments, R₁ and R₂ are each independently from eachother selected from H, straight C₁-C₅ alkyl, L1 is absent and L2 isselected from —(CH₂)—S—, —(CH₂)—O—, —(CH₂)—; R₃ and R₄ are eachindependently from each other be absent or selected from a ring systemcontaining five to 15 atoms, each optionally substituted with at leastone of straight or branched C₁-C₅ alkyl, —C(O)—CH₃, —C(O)—O—CH₃, halide,CF₃, nitro, amide, or R₅, R₅ is an a ring system containing five totwelve atoms optionally substituted by at least one halide or straightC₁-C₅ alkyl or branched C₁-C₅ alkyl.

In some other embodiments, R₁ and R₂ are each independently from eachother selected from H, straight C₁-C₅ alkyl,

L1 is absent and L2 is selected from —(CH₂)—S—, —(CH₂)—O—, —(CH₂)—;

R₃ is a bicyclic ring and R₄ is an aryl or a heteroaryl.

In yet some alternative particular embodiments of the SMC modulator foruse in accordance with the invention, the compound of Formula XI, mayprovide a compound having the general formula (XIII):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H, a ringsystem containing five to seven atoms, each optionally substituted by atleast one of halide, amide, amine, nitro;

L1 and L2 if present are each independently may be absent or from eachother selected from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—, —(CH₂)—,—(CH₂)—O—, —NH—(CH₂)— each independently from each other optionallysubstituted with C₁-C₅ alkyl, a ring system containing five to sevenatoms optionally substituted with C₃-C₅ alkyl;

R₃ and R₄ are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight C₁-C₅ alkyl, (═O), (═S), or R₅, R₅ is an aring system containing five to seven atoms optionally substituted by atleast one halide or straight C₁-C₅ alkyl.

In more specific embodiments for the compound of Formula XIII, at leastone of R₁ and R₂ is C₅-C₇ saturated cycloalkyl, C₅-C₇ saturatedcycloalkylene, C₅-C₇ aryl or C₅-C₇ arylene, L1 may be absent or selectedfrom —CH₂—CH₂—C(O)—N—CH₂—C(O)—NH—CH₂—, —CH₂—, each optionallysubstituted with ethyl, methyl, an aryl optionally substituted with amethyl or ethyl, L2 may be absent or may be selected from CH₂, at leastone of R₃ and R₄ may be C₂-C₁₂ heterocycloalkyl ring, C₂-C₁₂ heteroarylor C₂-C₁₂ heteroarylene. In some embodiments, the heteroatom may be N,O, S.

In still further some embodiments, at least one of R₁ and R₂ is selectedfrom the group consisting of cyclopentane or thiazole, L1 is absent or—CH₂—CH₂—C(O)—N—CH₂—C(O)—NH—CH₂—, substituted with an aryl optionallysubstituted with a methyl, L2 is absent or selected from CH₂, at leastone of R₃ and R₄ is selected from the group consisting of piperidine,piperazine, quinoline, isoquinoline, phthalazine, tetrahydro-quinoline,pyridine, tetrahydro-quinazoline substituted with —C(O)—CH₃.

In some particular embodiments, specific and non-limiting examples ofthe SMC modulators for use of the invention, or pharmaceuticallyacceptable salts or hydrates of the compounds of Formula XI, or in somefurther specific embodiments, pharmaceutically acceptable salts orhydrates of the compounds of Formula XIII may include:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide(Designated herein as SMC#33).

In yet another alternative, the compound may be

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester (Designated herein as SMC#30).

In yet some further embodiments, specific examples of compounds orpharmaceutically acceptable salts or hydrates of the compounds ofFormula I that may be used by the invention include, without limitation:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide(Designated herein as SMC#33);

1-(2,6-dimethylpiperidin-1-yl)-2-[[5-(quinolin-8-yloxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]ethanone(Designated herein as SMC #34);

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester (Designated herein as SMC#30);

ethyl 4-[[3-(4-acetylpiperazin-1-yl)sulfonylbenzoyl]amino]benzoate(Designated herein as SMC #23);

N-[2-(dimethylamino)-2-thiophen-2-ylethyl]-4-[ethyl(phenyl)sulfamoyl]benzamide (Designated herein as SMC #24);

10-(3-Chloro-benzyl)-8-(4-methyl-piperazine-1-carbonyl)-5,5-dioxo-5,10-dihydro-5λ6-dibenzo[b,f][1,4]thiazepin-11-one(Designated herein as SMC #26);

2-(4-Morpholin-4-yl-6-phenylamino-[1,3,5]triazin-2-ylsulfanyl)-N-(3-nitro-phenyl)-acetamide(Designated herein as SMC #15);

3-(benzylsulfamoyl)-N-[2-(2,6-difluoroanilino)-2-oxoethyl]-N-ethylbenzamide(Designated herein as SMC #25);

2-[4-[3-(dimethylsulfamoyl)-4-methylphenyl]-1-oxophthalazin-2-yl]-N,N-dimethylacetamide(Designated herein as SMC #16);

N-(2,5-difluorophenyl)-2-[2-[1-(4-methylphenyl)sulfonylpiperidine-3-carbonyl]hydrazinyl]acetamide(Designated herein as SMC #21);

2-fluoro-N-[4-[[2-[(4-methyl-5-pyridin-4-yl-1,2,4-triazol-3-yl)sulfanyl]acetyl]amino]phenyl]benzamide(Designated herein as SMC #31);

2-[2-(naphthalen-2-ylamino)-2-oxoethyl]sulfanyl-N-[(4-propan-2-ylphenyl)methyl]acetamide(Designated herein as SMC #17);

1-(3-chloro-4-fluorophenyl)-3-[[1-[(2,5-dimethylphenyl)methyl]piperidin-4-yl]methyl]urea(Designated herein as SMC #28).

In some alterative embodiments, the compound of the invention may be anycompound defined by having general formula (I) above provided that thecompound is not any one of the compounds detailed below, specifically:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide(Designated herein as SMC #33);

1-(2,6-dimethylpiperidin-1-yl)-2-[[5-(quinolin-8-yloxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]ethanone(Designated herein as SMC #34);

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester (Designated herein as SMC #30);

ethyl 4-[[3-(4-acetylpiperazin-1-yl)sulfonylbenzoyl]amino]benzoate(Designated herein as SMC #23);

N-[2-(dimethylamino)-2-thiophen-2-ylethyl]-4-[ethyl(phenyl)sulfamoyl]benzamide (Designated herein as SMC #24);

10-(3-Chloro-benzyl)-8-(4-methyl-piperazine-1-carbonyl)-5,5-dioxo-5,10-dihydro-5λ6-dibenzo[b,f][1,4]thiazepin-11-one(Designated herein as SMC #26);

2-(4-Morpholin-4-yl-6-phenylamino-[1,3,5]triazin-2-ylsulfanyl)-N-(3-nitro-phenyl)-acetamide(Designated herein as SMC #15);

3-(benzylsulfamoyl)-N-[2-(2,6-difluoroanilino)-2-oxoethyl]-N-ethylbenzamide(Designated herein as SMC #25);

2-[4-[3-(dimethylsulfamoyl)-4-methylphenyl]-1-oxophthalazin-2-yl]-N,N-dimethylacetamideDesignated herein as SMC #16);

N-(2,5-difluorophenyl)-2-[2-[1-(4-methylphenyl)sulfonylpiperidine-3-carbonyl]hydrazinyl]acetamide(Designated herein as SMC #21);

2-fluoro-N-[4-[[2-[(4-methyl-5-pyridin-4-yl-1,2,4-triazol-3-yl)sulfanyl]acetyl]amino]phenyl]benzamide(Designated herein as SMC #31);

2-[2-(naphthalen-2-ylamino)-2-oxoethyl]sulfanyl-N-[(4-propan-2-ylphenyl)methyl]acetamide(Designated herein as SMC #17);

1-(3-chloro-4-fluorophenyl)-3-[[1-[(2,5-dimethylphenyl)methyl]piperidin-4-yl]methyl]urea(Designated herein as SMC #28);

Still further, in some embodiments, the SMC isN′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamidehaving a structure:

(denoted herein as SMC #33)

In some embodiments, the SMC is1-(2,6-dimethylpiperidin-1-yl)-2-[[5-(quinolin-8-yloxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]ethanonehaving a structure

In some embodiments, the SMC is ethyl4-[[3-(4-acetylpiperazin-1-yl)sulfonylbenzoyl]amino]benzoate having astructure

(denoted herein as SMC #23);

In some embodiments, the SMC is10-(3-Chloro-benzyl)-8-(4-methyl-piperazine-1-carbonyl)-5,5-dioxo-5,10-dihydro-5λ6-dibenzo[b,f][1,4]thiazepin-11-onehaving a structure

(denoted herein as SMC #26);

In accordance with some other aspects, the present disclosure provides acompound having the general formula (V):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

each one of X, Y, Z, V, W, T and S may be selected from N, NH and C,

R₆ and R₇ are the same or are different and are independently selectedfrom each other may be L3-R₅,

L3 may be selected from —(CH₂)_(n), —NH—C(O) and C(O)—NH, S(O)₂, C(O),

n is an integer between 0 to 5;

R₅ may be selected from a ring system containing five to twelve atoms,each optionally substituted by at least one of straight or branchedC₁-C₅ alkyl, halide, hydroxyl, ester, ether, amide, nitro and hydroxyl,CF₃.

In accordance with some embodiments, the compound having the generalformula (V) may be represented by the general formula (VI):

wherein each one of X, Y, Z, V, W, T, S, R₆ and R₇ are as defined informula (V).

In accordance with some embodiments, the compound having the generalformula (V) may be represented by the general formula (VII):

wherein each one of X, Y, Z, V, W, T, S, R₆ and R₇ are as defined informula (V).

In accordance with some embodiments, the compound having the generalformula (V) may be represented by the general formula (VIII):

wherein X, Y, Z, W and T may be selected from N and V is selected fromNH;

L3 may be selected from —(CH₂)—, and —S(O)₂;

R₈ may be an aryl optionally substituted by at least one halide or CF₃.

In some embodiments, specific examples of compounds or pharmaceuticallyacceptable salts or hydrates of the compounds of Formula V include,without limitation:

3-[(3-fluorophenyl)methyl]-5-[1-[2-(trifluoromethyl)phenyl]sulfonylpiperidin-4-yl]-2H-triazolo[4,5-d]pyrimidin-7-one

(Designated herein as SMC #32);

In some alterative embodiments, the compound of the invention may be anycompound defined by having general formula (V) above provided that thecompound is not

3-[(3-fluorophenyl)methyl]-5-[1-[2-(trifluoromethyl)phenyl]sulfonylpiperidin-4-yl]-2H-triazolo[4,5-d]pyrimidin-7-one

(Designated herein as SMC #32);

In accordance with some other aspects, the present disclosure provides acompound having the general formula (IX):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

L4 may be absent or may be selected from —(CH₂)_(n)—, —S—(CH₂)_(n)—,—(CH₂)_(n)—S—; n is an integer between 0 and 5;

R₁₀ and R₁₁ are each independently from each other absent or selectedfrom a ring system containing five to twelve atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), —C(O)—CH₃, —C(O)—O—CH₃, halide, nitro, NH₂.

In some embodiments, L4 is selected from —S—(CH₂)—. In some otherembodiments, R₁₀ and R₁₁ are each independently from each other absentor selected from triazine, piperidine each optionally substituted withat least one NH₂.

In some embodiments, specific examples of compounds or pharmaceuticallyacceptable salts or hydrates of the compounds of Formula IX include,without limitation:

8-[(4-amino-6-piperidin-1-yl-1,3,5-triazin-2-yl)methylsulfanyl]-7-ethyl-1,3-dimethylpurine-2,6-dione

(Designated herein as SMC #18).

In yet some alterative embodiments, the compound of the invention may beany compound defined by having general formula (IX) above provided thatthe compound is not

8-[(4-amino-6-piperidin-1-yl-1,3,5-triazin-2-yl)methylsulfanyl]-7-ethyl-1,3-dimethylpurine-2,6-dione(Designated herein as SMC #18).

In accordance with some other aspects, the present disclosure provides acompound having the general formula (X):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁₃ and R₁₄ are each independently from each other absent or selectedfrom a ring system containing five to twelve atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, C₁-C₅alkoxy, (═O), (═S), —C(O)—CH₃, —C(O)—O—CH₃, halide, nitro, NH₂,NH—C(O)—CH₃.

In some other embodiments, R₁₀ and R₁₁ are each independently from eachother absent or selected from aryl each optionally substituted with atleast one OCH, NH—C(O)—CH₃.

In some embodiments, specific examples of compounds or pharmaceuticallyacceptable salts or hydrates of the compounds of Formula X include,without limitation:

N-[4-[3-(2,4-dimethoxyphenyl)-4-oxo-1,2-dihydroquinazolin-2-yl]phenyl]acetamide

(Designated herein as SMC #22).

In yet some alterative embodiments, the compound of the invention may beany compound defined by having general formula (X) above provided thatthe compound is not

N-[4-[3-(2,4-dimethoxyphenyl)-4-oxo-1,2-dihydroquinazolin-2-yl]phenyl]acetamide

(Designated herein as SMC #22).

Still further, in some embodiments, the compound for use in accordancewith the uses of the invention may be the SMC#6, specifically,N-[(2R,4R,6S)-2-(4-chlorophenyl)-6-(1-methylbenzotriazol-5-yl)oxan-4-yl]acetamide.

In yet some alternative embodiments, the invention provides the uses ofany of the SMCs disclosed herein, provided that said SMC is not SMC#6,specifically,N-[(2R,4R,6S)-2-(4-chlorophenyl)-6-(1-methylbenzotriazol-5-yl)oxan-4-yl]acetamide.

The term “alkyl” as used herein refers to a linear, branched saturatedhydrocarbon having from 1 to 20 carbon atoms. The term “C₁-C₁₂ alkyl” or“C₁-C₁₂ alkylene” refers to a linear (straight), branched saturatedhydrocarbon having from 1 to 12 carbon atoms, in some embodiments,contain from 2 to 8 carbons, in yet some embodiments from 2 to 5carbons, in yet some further embodiments, from 1 to 3 carbon atoms. Itshould be noted that alkyl refers to an alkyl end chain and alkylenerefers to a middle chain alkyl. Representative C₁-C₁₂ alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, sec-butyl, iso-butyl, tert-butyl, cyclobutyl,pentyl, iso-pentyl, neo-pentyl, tert-pentyl, cyclopentyl, hexyl,cyclohexyl, heptyl, cycloheptyl, octyl, sec-octyl (1-methylheptyl), andcyclooctyl.

The term “C₁-C₁₂ haloalkyl” as used herein refers to a C₁-C₁₂ alkyl asdefined above, with one or more hydrogens substituted by halogen atoms.

The term “alkenyl” as used herein refers to a linear (straight),branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and atleast one carbon-carbon double bond. The term “C₂-C₁₂ alkenyl” or“C₂-C₁₂ alkenylene” as used herein refers to a linear, branchedunsaturated hydrocarbon having from 2 to 12 carbon atoms and at leastone carbon-carbon double bond, in some embodiments from 3 to 8 carbons,in yet some further embodiments, from 3 to 5 carbon atoms and at leastone double bond. It should be noted that alkenyl refers to an alkyl endchain and alkenylene refers to a middle chain alkyl.

The term “C₂-C₁₂ haloalkenyl” as used herein refers to a C₂-C₁₂alkenylas defined above, with one or more hydrogens substituted by halogenatoms.

The term “alkynyl” as used herein refers to a linear, branchedunsaturated hydrocarbon having from 2 to 20 carbon atoms and at leastone carbon-carbon triple bond. The term “C₂-C₁₂ alkynyl” or “C₂-C₁₂alkynylene” as used herein refers to a linear, branched unsaturatedhydrocarbon having from 2 to 12 carbon atoms in certain embodiments,from 3 to 8 carbons, and at least one triple bond (at least onecarbon-carbon triple bond). It should be noted that alkynyl refers to analkyl end chain and alkynylene refers to a middle chain alkyl.

The term “C₂-C₁₂ haloalkynyl” as used herein refers to a C₂-C₁₂ alkynylas defined above, with one or more hydrogens substituted by halogenatoms.

As used herein “alkoxy” refers to an alkyl group bonded to an oxygenatom. Similarly, the term “C₁-C₁₂ alkoxyl” as used herein refers to aC₁-C₁₂ alkyl group linked to an oxygen. At times, the alkyl group mayinclude one to twelve carbon atoms, at times between one to eight carbonatoms, at times one to five carbon atoms and at times one to threecarbon atoms. Representative examples are methoxy, ethoxy, n-propoxy,isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy,hexoxy, isohexoxy and the like. In certain embodiments, the alkoxy isethoxy.

The term “halogen” (halo or halide) refers to F, C₁, Br or I.

As used herein, a ring system containing five to twelve atoms refers toa mono- or multi-cyclic ring system having 5 to 12 atoms. The ringsystem containing five to twelve atoms may be saturated, unsaturated oraromatic rings and the like including for example cycloalkyl,heterocycloalkyl, aryl, arylene, aromatic, heteroaromatic rings. A ringsystem containing five to twelve atoms may contain two rings (bicyclic,etc.), for example aromatic rings and in such case the aromatic rings ofthe aryl group may be joined at a single point (e.g., biphenyl), orfused (e.g., naphthyl). In some embodiments, a ring system containingfive to twelve atoms is a carbocyclic ring or heterocyclic ring. Theterm “carbocyclic ring” refers to cyclic compounds containing onlycarbon atoms. The carbocyclic ring may be optionally substituted by oneor more substituents, and may be saturated, unsaturated or aromatic. Theterm “heterocyclic ring” refers to cyclic compounds where one or morecarbons are substituted by heteroatoms. Exemplary heteroatoms include,but not limited to, nitrogen, sulfur, and oxygen. The heterocyclic ringmay be optionally substituted, and may be saturated, unsaturated oraromatic. The term “saturated” as used herein means that the compounddoes not contain double or triple bonds. The term “unsaturated” as usedherein means that the compound contains at least one double or triplebond. The term “aromatic” as used herein means that the compoundcontains alternating double and single bonds.

As used herein, “aryl” refers to aromatic ring systems having between 5to 12 atoms. Unless otherwise specifically defined, the term “aryl”refers to cyclic, aromatic hydrocarbon groups that have 1 to 2 aromaticrings, including monocyclic or bicyclic groups having between 5 to 12atoms. Non-limiting examples include phenyl, biphenyl or naphthyl. Thearyl group may be optionally substituted by one or more substituents,e.g., 1 to 5 substituents, at any point of attachment. The substituentscan themselves be optionally substituted. As used herein, “C₅-C₁₂aromatic” refers to aromatic ring systems having 5 to 12 carbon atoms,such as phenyl, naphthalene and the like.

As used herein, the term “heteroaryl” refers to aryls as defined abovewhere one or more carbons are substituted by heteroatoms. Exemplaryheteroatoms include, but not limited to, nitrogen, sulfur, and oxygen.As used herein, “heteroaromatic” refers to refers to a monocyclic ormulti-cyclic (fused) aromatic ring system, where one or more of theatoms in the ring system is a heteroatom, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen or sulfur. Theterm “heteroaromatic” used interchangeably with the term “heteroaryl”denotes a heterocyclic aromatic ring systems containing 5 to 12 atoms,with at least one, preferably two carbon atoms and one or moreheteroatoms selected from nitrogen, oxygen and sulfur. Non-limitingexamples include furan, thipohene, pyrrole, oxazole, oxadiazole,thiazole, imidazole, pyrazole, isoxazole, thiazolem benzofurna, indole,benzothiophene, benzoimidazole, indazole, benzoxazole, benzoisoxazole,benzothiazole, isobenzfuran, isoidole, purine, pyridine, pyrazine,pyrimidine, pyrisazine, quinoline, quinozaline, quinazoline,isoquinoline, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl,isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyranyl,pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl,1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl,thiadiazinyl, [1,3,4]thiadiazole, thiadiazole, indolyl, isoindolyl,benzofuryl, benzothienyl, indazolyl, benzimidazolyl, benzothiazolyl,benzoisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl,quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl,pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl and the like.

As used herein, “C₅-C₁₂ saturated cycloalkyl” refers to a saturatedmono- or multi-cyclic ring system having 5 to 12 carbon atoms,preferably having 5 to 7 carbon atoms. Example of “C₅-C₁₂ cycloalkyl”groups include, but are not limited to cyclopentyl, cyclohexyl andcycloheptyl.

As used herein, “heterocycloalkyl” or “heterocyclyl” or the term“heterocyclic” refers to a monocyclic or multi-cyclic non-aromatic ringsystem having 5 to 12 members, preferably having 5 to 7 carbon atoms,where one or more, in certain embodiments, 1 to 3, of the atoms in thering system is a heteroatom, that is, an element other than carbon,including but not limited to, nitrogen, oxygen or sulfur. Examples of“heteroalkyl” include, but are not limited to, pyran, 1,4-dioxane,1,3-dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran,tetrahydrothiophene, and the like. The term heterocycloalkyl” alsoencompasses non-aromatic ring being unsaturated or having one or moredegrees of unsaturation containing one or more heteroatomicsubstitutions selected from S, SO, SO₂, O, or N. “heterocyclic” ring(s)or cycloalkyl ring(s). Examples of “heterocyclic” include, but are notlimited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane,piperidine, pyrrolidine, morpholine, tetrahydrothiopyran,tetrahydrothiophene, and the like.

The term “bicyclic ring” or “bicyclic group” as used herein refers tosystem or group that features two joined rings and encompasses acarbocyclic (all of the ring atoms are carbons), or heterocyclic (therings atoms consist of at least two different elements). The bicyclicgroup can be aromatic, aliphatic or a combination of aliphatic andaromatic. The bicyclic group for example can be any one of the following(i) the two rings share only one single atom, (ii) fused bicycliccompounds, two rings share two adjacent atoms, (iii) bridged bicycliccompounds the two rings share three or more atoms, separating the twobridgehead atoms by a bridge containing at least one atom. The bicyclicgroup can be optionally substituted as detailed herein.

Non-limiting example of bicyclic group include benzofuran,isobenzofuran, isoindole, benzothiophene, benzo[c]thiophene,benzimidazole, indazole, benzoxazole, benzisoxazole, benzothiazole,naphthalene, quinoline, isoquinoline, quinoxaline, quinazoline,cinnoline, phthalazine.

The term “N-containing group” is used herein a chemical group containinga nitrogen atom for example as amino group. The term “amino” as usedherein encompass primary, secondary, tertiary or quaternary amines wherethe point of attachment is through the nitrogen atom which issubstituted. For example, the “N-containing group” include N, NH, NH₂,tertiary amine (tertiary alkyl amine), quaternary ammonium (quaternaryalkyl ammonium). The nitrogen atom may be substituted with alkyl. Incase of a tertiary amine or quaternary amines, the substituent may bethe same or may be different.

The term “bond” as used herein denotes a covalent bond. The bond may bebetween two similar atoms or between different atoms. Non-limitingexamples include C—C, C—S, C—O, C—N. S—O, S—N, N—O and the like. Itshould be noted that a bond as defined above, for example, C—Sencompasses both C—S and S—C and this holds for the bonds as definedherein.

The term “optionally substituted” refers to substitution with the namedsubstituent or substituents, multiple degrees of substitution beingallowed unless otherwise stated. The term substituted as used hereinmeans that the compounds may contain one or more substituents,including, but not limited to, optionally substituted OH, CF₃, halogen,C(═O), —COOH, —NH₂, CN, alkyl, alkenyl, alkynyl, alkylene, straightalkenylene, alkynylene, haloalkyl, haloalkenyl, haloalkynyl, alkoxy,carboxyl, halogen, ring system including five to twelve atoms, aromaticor heteroaromatic ring, C(═O)— alkyl.

It should be noted that the carbon number, as used herein, refers to thecarbon backbone and carbon branching, but does not include carbon atomsof the substituents, such as alkoxy substitutions and the like.

The invention provides new SMC that act as WASp modulators. A smallmolecule in the context of the present disclosure refers to a lowmolecular weight organic compound, having a molecular weight lower than900 Daltons. In accordance with the present disclosure, when referringto a small molecule it includes also crystalline and amorphous forms ofthose compounds, including, for example, polymorphs, pseudopolymorphs,solvates, hydrates, unsolvated polymorphs (including anhydrates),conformational polymorphs, and amorphous forms of the compounds, as wellas mixtures thereof. “Crystalline form” or “polymorph,” as used hereininclude all crystalline and amorphous forms of a small molecule,including, for example, polymorphs, pseudopolymorphs, solvates,hydrates, unsolvated polymorphs (including anhydrates), conformationalpolymorphs, and amorphous forms, as well as mixtures thereof, unless aparticular crystalline or amorphous form is referred to.

In accordance with the present disclosure, the term “small molecule” mayinclude pharmaceutically acceptable forms of the recited compounds,including chelates, non-covalent complexes, prodrugs, and mixturesthereof. Further and in accordance with the preset disclosure, the term“small molecule” includes also pharmaceutically acceptable forms of aparticular molecule and as such the term small molecule also encompassespharmaceutically acceptable salts.

As detailed above, in accordance with the first aspect, the presentdisclosure provides small molecule compounds that bind to theN′-terminus of WASp for use in modulating WASp degradation and/orstability in a cell. More specifically, in some embodiments, the SMCmodulator of the invention specifically binds to WASp degradationpocket. In more specific embodiments, WASp degradation pocket is locatedat the N-terminal WASp-homology-1 (WH1) domain of WASp. Morespecifically, this pocked comprise at least one of lysine residues 76and 81. Thus, in more specific embodiments, the SMCs of the inventionare specifically directed to the N-terminal WH1 domain of WASp.

As used herein, WASp (Wiskott—Aldrich syndrome protein) is a 502-aminoacid protein encoded by a gene located at the short arm of the Xchromosome (Xp11.22-p11.23.1). WASp serves as an adaptor protein thatfacilitates actin cytoskeletal rearrangements that are essential fornormal immune cell responses. More specifically, WASp is a member of adistinct family of proteins that participate in the transduction ofsignals from the cell surface to the actin cytoskeleton. This family ischaracterized by a C-terminal tripartite domain containing a commonactin monomer-binding motif, WASp-homology domain 2 (WH2) or verprolinhomology domain (V), and a central-acidic (CA) region which is capableof activating the actin related protein (Arp)2/3 complex, a potentnucleator of actin polymerization. The activities of WASp family membersare normally tightly controlled within a cell, allowing for both spatialand temporal regulation of actin polymerization. WASp is regulated byseveral mechanisms including the adoption of an auto inhibitedconformation in which the VCA domain forms a hydrophobic interactionwith the GTPase binding domain (GBD, residues 230-288) and adjacentC-terminal residues. Binding of GTP-loaded Cdc42 andphosphatidylinositol 4, 5-bisphosphate (PIP2) appears to cooperativelydisrupt this interaction, thereby freeing the C-terminus for binding tothe Arp2/3 complex. Serine and tyrosine phosphorylation of WASp alsoacts to directly regulate its activity, although until recently this hasbeen relatively unexplored, particularly in vivo. The N-terminus ofWASp/N-WASp contains an Ena/VASP homology 1 (EVH1), which binds thewidely expressed verprolin homologue, WIP (WASp interacting protein).The majority of WASp in cells is complexed with WIP, and the WIP/WASpinteraction is important for WASp activation by Cdc42 through Toca-1.WIP is a molecular chaperone crucial for the stability of WASp, whichotherwise is susceptible to proteolytic cleavage through calpain andpossibly the 26S ubiquitin proteasome systems.

As used herein, WASp is a protein exclusively expressed in hematopoieticcells. In some embodiments, said WASp may be a human WASp. In morespecific embodiments, said human WAS protein may comprise the amino acidsequence as denoted by Accession number P42768 (CCDS: CCDS14303.1). Inmore specific embodiments, the amino acid sequence of WASp is as denotedby SEQ ID NO. 1. In some specific embodiments said WASp is encoded bythe cDNA of Accession number NM_000377. In more specific embodiments,said cDNA sequence of WASp is denoted by SEQ ID NO. 2.

As demonstrated in FIG. 13, the SMC modulators of the invention,specifically, any of the modulators disclosed by the invention inconnection with any aspects thereof, may enhance and stabilize theprotective interaction between WASp and WIP. Therefore, in certainembodiments, the SMC modulators of the invention may act as enhancersand stabilizers of the WASp and WIP interaction. In yet some furtheralternative embodiments, the compounds of the invention may act as WIPmimetic compounds, thereby protecting WASp from proteolysis.

As shown in FIG. 7, by binding to the degradation pocket in WASp, theSMC modulators of the invention modulate WASp ubiquitylation. Thus, insome embodiments, binding of the SMC modulators of the invention(specifically, any of the modulators disclosed by the invention inconnection with any aspects thereof), to WASp may lead to modulation ofWASp ubiquitylation in a cell. It should be noted that “modulation” asused herein encompasses either “inhibition of ubiquitylation, oralternatively, enhancement”.

More specifically, the terms “inhibition”, “moderation”, “reduction” or“attenuation” as referred to herein, relate to the retardation,restraining or reduction of WASp ubiquitylation by any one of about 1%to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10%to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30%to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50%to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75%to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95%to 99%, or about 99% to 99.9%. Alternatively, the terms “increase”,“augmentation” and “enhancement” as used herein relate to the act ofbecoming progressively greater in size, amount, number, or intensity.Particularly, an increase of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%,400%, 500%, 600%, 70%, 800%, 900%, 1000% or more of WASp ubiquitylationas compared to a suitable control. It should be appreciated that 10%,50%, 120%, 500%, etc., are interchangeable with “fold change” values,i.e., 0.1, 0.5, 1.2, 5, etc., respectively. 10%, 50%, 120%, 500%, etc.,are interchangeable with “fold change” values, i.e., 0.1, 0.5, 1.2, 5,etc., respectively. Therefore, the term increase refers to an increaseof about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 folds or more.

More specifically, the invention provides different SMCs that followingbinding to WASp, specifically mask its degradation sites at lysineresidues 76 and 81, located in a pocket at the N′-terminalWASp-homology-1 (WH1) domain of WASp. Thus, in some embodiments, SMCbinding to WASp induces modulation of ubiquitin proteasome-mediateddegradation of WASp in a cell.

“Ubiquitylation-dependent proteosomal degradation” as used herein, is aclassical protein degradation pathway within a cell that includeshydrolysis by the proteasome after conjugation of polyubiquitin chains.Before the degradation by the proteasome, a protein undergoesphosphorylation-dependent ubiquitylation. The ubiquitin-proteasomesystem plays important role in several biological processes such asantigen presentation, endocytosis and cell stress response andrepresents one of the most important degradation systems in the cell.Misfolded and unfolded proteins are targets for degradation byproteasome in order to maintain cell integrity and survival. Proteinubiquitylation usually requires three processes involvingubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2s) andubiquitin ligases (E3s).

As clearly demonstrated in EXAMPLE 3, the prevention of degradation ofWASp by the SMC modulators of the invention refers to theubiquitylation-dependent proteosomal degradation mechanism of WASp inhematopoietic cells, specifically, in platelets, megakaryocytes andlymphocytes.

Thus, in some particular embodiments for the SMC modulator for use inaccordance with the invention, any of the described SMC modulatorcompounds, may attenuate WASp ubiquitylation thereby reducing ubiquitinproteasome-mediated degradation of WASp in a cell.

In more specific embodiments of the SMC modulator of the invention foruse, the reduced degradation of WASp caused by the SMC modulator/s mayrestore, enhance or extend at least one of WASp levels and function inthe cell.

It should be understood that where referring to levels of WASp, theinvention further encompasses WASp expression and/or stability.“Expression”, as used herein generally refers to the process by whichgene-encoded information is converted into the structures present andoperating in the cell. Therefore, according to the invention“expression” of a gene, specifically, may refer to transcription into apolynucleotide, translation into a protein, or even posttranslationalmodification of the protein. Protein stability, as used herein, refersto the physical (thermodynamic) stability, and chemical stability of theprotein and relates to the net balance of forces, which determinewhether a protein will be in its native folded conformation or adenatured state. More specifically, the levels of proteins within cellsare determined not only by rates of synthesis as discussed above, butalso by rates of degradation and the half-lives of proteins within cellsthat vary widely, from minutes to several days. In eukaryotic cells, twomajor pathways mediate protein degradation, the ubiquitin-proteasomepathway, mentioned herein before, and lysosomal proteolysis.

According to some embodiments, wherein indicate “increasing” or“enhancing” the expression or the levels of WASp, it is meant that suchincrease or enhancement may be an increase or elevation of between about10% to 100% of the expression and/or stability of WASp. The terms“increase”, “augmentation” and “enhancement” as used herein relate tothe act of becoming progressively greater in size, amount, number, orintensity. Particularly, an increase of 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%,300%, 400%, 500%, 600%, 70%, 800%, 900%, 1000% or more of the expressionas compared to a suitable control. It should be further noted thatincrease or elevation may be also an increase of about 2 to 10⁶ folds ormore. Still further, it should be appreciated that the increase of thelevels or expression of said WASp may be either in translation or thestability of said WASp. With regards to the above, it is to beunderstood that, where provided, percentage values such as, for example,10%, 50%, 120%, 500%, etc., are interchangeable with “fold change”values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. Therefore, the termincrease refers to an increase of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000 folds or more.

As noted above, WASp is a multifunction protein, acting as an adaptorprotein that facilitates actin cytoskeletal rearrangements in responseto signals arising at the cell membrane, which are essential for normalimmune cell responses. In particular its function includes actinpolymerization, sustaining of the immunological synapse, endocytosis,calcium flux, NFAT gene transcription, cellular activation andproliferation.

Moreover, WASp emerges as an important platform for regulating actinpolymerization through activation of the Arp2/3 complex, which is a keyfor cytoskeletal reorganization. It is therefore not surprising thatlack of WASp results in a wide range of defects of cellular functioninvolving all hematopoietic cell lineages.

It is thus appreciated that the SMC modulators of the invention mayrestore WASp function, or phenotype in a cell. The term “phenotype ofWASp” as used herein defines the extent of expression of mRNA of WASp,as well as an amount and function of the WAS protein, wherein“restoration of WASp phenotype” means a return of the expression, amountand function to normal levels.

Therefore, in some embodiments, the SMC modulators of the inventionprotect from WASp degradation and thereby restore the phenotype of WASpin a cell. Specifically the SMCs of the invention restore theexpression, amount and function of WASp within a cell. Restore, as usedherein in connection with the levels and function of WASp, refers toreinstate, bring back, reinstitute, re-impose, reinstall, reestablish,repair, renovate, recover, elevate, improve, reconstitute, revive,renew, rescue or refresh WAPs levels and/or function, specifically, in acell displaying abnormal levels of WASp, by the SMCs of the invention inabout 5% to 100% or more of the normal WASp levels or function ascompared to cells displaying abnormal WASp levels that are not treatedwith the SIMCs of the invention. More specifically, the SMCs of theinvention restore about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,200%, 300%, 400%, 500%, 600%, 70%, 800%, 900%, 1000% or more of WASplevels or function of normal cells as compared to cells displayingabnormal WASp levels that were not treated by the SMCs of the invention.

In certain embodiments, the SMCs of the invention may restore WASplevels and function by attenuating its degradation in hematopoieticcells, for example, leukocytes, platelets and megakaryocytes. Asdetailed in EXAMPLES 2 and 3, the specific SMC modulators of theinvention decreased WASp degradation and thereby restored WASpexpression in platelets, peripheral blood mononuclear cells (FIG. 4,FIG. 5, FIG. 6) and T cell lymphocytes (FIG. 5). Retention of WASplevels results in restoration of WASp-mediated cellular function/s.

In some specific embodiment, the cell treated by the SMC modulator/sused in accordance with the invention, may be a hematopoietic cell, morespecifically, a non-erythroid hematopoietic cell.

In more specific embodiments, the SMC modulators used by the inventionreduce WASp degradation in a non-erythroid hematopoietic cell that maybe in some embodiments, at least one of a lymphocyte, platelet,megakaryocyte, granulocyte and monocyte. Thus, in certain specificembodiments, the SMC modulators of the invention may modulate, andspecifically, reduce, the WASp ubiquitylation in at least one oflymphocyte, platelet, megakaryocyte, granulocyte and monocyte.

“Hematopoietic cells” are cellular blood components all derived fromhematopoietic stem cells in the bone marrow. It should be appreciatedthat in certain embodiments, hematopoietic cells as used herein includecells of the myeloid and the lymphoid lineages of blood cells. Morespecifically, myeloid cells include monocytes, (macrophages anddendritic cells (DCs)), granulocytes (neutrophils), basophils,eosinophils, erythrocytes, and megakaryocytes or platelets. The Lymphoidcells include T cells, B cells, and natural killer (NK) cells. Thus, incertain embodiments, the cells treated by the SMC modulators of theinvention may be any hematopoietic cell described herein. Generally,blood cells are divided into three lineages: red blood cells (erythroidcells) which are the oxygen carrying, white blood cells (leukocytes,that are further subdivided into granulocytes, monocytes andlymphocytes) and platelets (thrombocytes).

In certain embodiments, the hematopoietic cells treated by the SMCmodulators of the invention may be non-erythroid hematopoietic cells.The term “non-erythroid hematopoietic cell” refers to the cells derivedfrom white blood cell precursors and from megakaryocytes and include atleast one of granulocytes (neutrophils, basophils, eosinophils),monocytes, lymphocytes, macrophages, dendritic cells and platelets.

In some embodiments, the cell is lymphocyte. In some other embodiments,the cell is T lymphocyte. In some further embodiments, the cell isplatelet. In some other embodiments, the cell is megakaryocyte.

Thus, as demonstrated in EXAMPLE 4 (FIG. 8-FIG. 11) addition of the SMCmodulators of the invention to lymphocytes, platelets and megakaryocytesnot only causes upregulation of WASp expression in these cells but alsoresults in an increase in a specific function of each one of these cellsas detailed herein below.

In some specific embodiments, the SMC modulator of the invention mayattenuate WASp ubiquitylation in lymphocyte/s, specifically, Tlymphocyte/s.

In yet some further specific embodiments, the SMC modulator/s of theinvention may attenuate WASp ubiquitylation in platelet/s.

Still further, the SMC modulator/s of the invention may attenuate WASpubiquitylation in megakaryocyte/s.

As indicated above and demonstrated in the Examples, attenuation of WASpubiquitylation in a cell restores the levels (FIGS. 4-6) and function(FIGS. 8-11) of WASp in a cell. In more specific embodiments, functionof WASp may result in at least one of cell activation, cellproliferation, cell migration, cell homing, cell spreading, cellaggregation, elevation in intracellular calcium concentration, celladhesion, phagocytosis, and cytolytic activity.

In some specific embodiments, the invention relates to restoring orexpending WASp levels and function in lymphocyte/s.

“Lymphocytes” are mononuclear nonphagocytic leukocytes found in theblood, lymph, and lymphoid tissues. They comprise the body'simmunologically competent cells and their precursors. They are dividedon the basis of ontogeny and function into two classes, B and Tlymphocytes, responsible for humoral and cellular immunity,respectively. Most are small lymphocytes 7-10 μm in diameter with around or slightly indented heterochromatic nucleus that almost fills theentire cell and a thin rim of basophilic cytoplasm that contains fewgranules. When “activated” by contact with antigen, small lymphocytesbegin macromolecular synthesis, the cytoplasm enlarges until the cellsare 10-30 μm in diameter, and the nucleus becomes less completelyheterochromatic; they are then referred to as large lymphocytes orlymphoblasts. These cells then proliferate and differentiate into B andT memory cells and into the various effector cell types: B cells intoplasma cells and T cells into helper, cytotoxic, and suppressor cells.

More specifically, lymphocytes as used herein include T cells, B cellsand NK cells. A “T cell” or “T lymphocyte” as used herein ischaracterized by the presence of a T-cell receptor (TCR) on the cellsurface. It should be noted that T-cells include helper T cells(“effector T cells” or “Th cells”), cytotoxic T cells (“Tc,” “CTL” or“killer T cell”), memory T cells, and regulatory T cells as well asnatural killer T cells, Mucosal associated invariants and Gamma delta Tcells.

More specifically, Thymocytes are hematopoietic progenitor cells presentin the thymus. Thymocytes are classified into a number of distinctmaturational stages based on the expression of cell surface markers. Theearliest thymocyte stage is the double negative (DN) stage (negative forboth CD4 and CD8), which more recently has been better described asLineage-negative, and which can be divided into four sub-stages. Thenext major stage is the double positive (DP) stage (positive for bothCD4 and CD8). The final stage in maturation is the single positive (SP)stage (positive for either CD4 or CD8).

B cells or B lymphocytes, develop from hematopoietic stem cells (HSC)that originate from bone marrow. 13 cells function in the humoralimmunity component of the adaptive immune system by secretingantibodies. B cells are characterized by the expression of B cellreceptor (BCR) that allows specificity to antigen, against which it willinitiate an antibody response. Additionally, B cells present antigen(classified as professional antigen-presenting cells (APCs)) and secretecytokines. Their development into B cells occurs in several stages, eachmarked by various gene expression patterns and immunoglobulin H chainand L chain gene loci arrangements, the latter due to B cells undergoingV(D)J recombination as they develop.

Natural killer cells or NK cells are a type of cytotoxic lymphocytecritical to the innate immune system. NK cells (belonging to the groupof innate lymphoid cells) are defined as large granular lymphocytes(LGL). The role NK cells play is analogous to that of cytotoxic T cellsin the vertebrate adaptive immune response. NK cells provide rapidresponses to viral-infected cells, acting at around three days afterinfection, and respond to tumor formation. Typically, immune cellsdetect major histocompatibility complex (MHC) presented on infected cellsurfaces, triggering cytokine release, causing lysis or apoptosis. NKcells are unique, however, as they have the ability to recognizestressed cells in the absence of antibodies and MHC, allowing for a muchfaster immune reaction. They were named “natural killers” because of theinitial notion that they do not require activation to kill cells thatare missing “self” markers of MHC class 1.

In some specific embodiment, the SMC modulator/s of the invention mayup-regulate lymphocyte activation. “Lymphocyte activation” as usedherein refers to stimulation of lymphocytes by specific antigen ornonspecific mitogens that result in synthesis of RNA, protein, and DNAand the production of cytokines. This process is followed byproliferation and differentiation of various effector and memory cells.Activation is accompanied by morphologic changes known as lymphocytetransformation, in which small, resting lymphocytes are transformed intolarge, active lymphocytes (lymphoblasts). As shown by the invention,lymphocyte activation may be evaluated by expression of activationmarkers. Thus, in some specific embodiments, the SMC modulator/s of theinvention may lead to lymphocyte/s activation as demonstrated byexpression of lymphocyte activation markers, specifically, CD69 andintegrins LFA-1), proliferation and migration.

“Lymphocyte proliferation” relates to the ability of lymphocytes torespond to mitogens, specific antigens, or allogenic cells. Morespecifically, cell proliferation is the process that results in anincrease of the number of cells, and is defined by the balance betweencell divisions and cell loss through cell death or differentiation.

As shown in FIG. 10, the SMCs of the invention enhance T lymphocytemigration. Thus, in yet some further embodiments, the SMC modulators ofthe invention induce lymphocyte migration.

“Lymphocyte Migration” refers to continually re-circulating lymphocytesbetween the blood and the tissues via the lymph. In order to maintainimmune surveillance, the majority of lymphocyte traffic occurs throughlymph nodes in vivo. Although a great deal of work has been done toelucidate the molecular mechanisms whereby lymphocytes leave the bloodand enter the lymph node, lymphocyte traffic also requires that thelymphocyte successfully transit extravascular tissue and enter the lymphfollowing transendothelial migration.

As part of lymphocyte activation and restoring the functions of WASp,the SMC modulators of the invention may also enhance, participate orinduce formation of immunological synapse (IS) by a T lymphocyte. Thus,the present invention may be further articulated from the point view ofa lymphocyte forming an immunological synapse (IS) as the target for thepresently conceived modulators. The IS model in relating to featuressuch as T cell membrane structure, T cell polarity, signaling pathways,and antigen-presenting cells (APC), provides a comprehensive view on Tcells maturation and activation. Originally the term ‘IS’ denoted acrucial junction between a T cell and APC at which T cell receptors(TCRs) interact with MHC molecules. Today however this term has beenexpended to include different types of immune cells, as well asnon-immune cells. Thus, the term ‘IS’ herein denotes a specificarrangement of molecules in an immune cell at the interface with anothercell. Molecules related to IS formation may include, although notlimited to, receptors, signaling molecules, cytoskeletal elements andcellular organelles. When referring to arrangement of said molecules ismeant, for example, accumulation of molecules in distinct regions withinan activating IS to form a supramolecular activation cluster (SMAC),which may be further segregated into peripheral (pSMAC) and central(cSMAC) zones. This term further encompasses other features, such asengagement of individual receptors, or involvement of microclusters ofcell-surface, and signaling molecules that support cell activation andmaturation of IS.

As indicated herein before, WASp-dependent immune cell functions includeactin polymerization, sustaining of the immunological synapse,endocytosis, calcium flux, NFAT gene transcription, cellular activationand proliferation. Thus, in certain embodiments, by restoring WASplevels and function, the SMC modulators of the invention may modulateactin polymerization. The term ‘actin’ has been articulated herein invarious contexts. In the above described aspects and embodiments it wasused to convey actin functionality and structural meaning. In mostgeneral terms, ‘actin’ is a ubiquitous globular protein that is one ofthe most highly-conserved proteins known.

Structurally, the term ‘actin’ refers to the two main states of actin:the G-actin—the globular monomeric form and the F-actin forming helicalpolymers. Both G- and F-actin are intrinsically flexible structures—afeature vital in actin's role as a dynamic filament network. In terms offunctionality, the F-actin polymers form microfilaments—polarintracellular ‘tracks’ for kinesin motor proteins, allowing thetransport of vesicles, organelles and other cargo. Further, actin is acomponent of the cytoskeleton and links to alpha-actinin, E-cadherin andbeta-catenin at adherent junctions. This gives mechanical support tocells and attaches them to each other and the extracellular matrix.Using energy from the hydrolysis of ATP, myofibrils undergo cyclicshortening through actin-myosin head interactions, which represents themechanics of muscle contraction. Finally, actin has a role in cellmotility through polymerization and depolymerization of fibrils.

In further embodiments, the SMC modulators of the invention mayupregulate platelet and/or megakaryocyte activation. In yet some otherembodiments, the SMC modulators of the invention may activate platelet/sand/or megakaryocyte/s as manifested by at least one of cell spreading,cell aggregation, elevation in intracellular calcium concentration, celladhesion, phagocytosis, and cytolytic activity.

A megakaryocyte is a large bone marrow cell with a lobulated nucleusresponsible for the production of blood thrombocytes (platelets), whichare necessary for normal blood clotting. Megakaryocytes are derived fromhematopoietic stem cell precursor cells in the bone marrow.

The “platelet/s” as used herein, are one of the key elements of humanblood, playing a central role in the process of thrombus formation. Themain function of platelets is the formation of mechanical plugs duringthe normal hemostatic response to the vessel wall injury. Platelets arederived from the megakaryocytes in the bone marrow. These megakaryocytesarise by a process of differentiation from the haemopoietic stem celland undergo fragmentation of their cytoplasm to produce platelets.Platelet production is under the control of humoral agents such asthrombopoietin. The platelet is an enucleate cell that beside nucleusincludes intracellular organelles in the cytoplasm. Resting plateletsare discoid and have a smooth, rippled surface. The platelet surface hasvarious receptors to which various stimulants (agonists) bind andthereby activate platelets producing changes within the platelet as wellas a change in platelet shape from discoid to spherical, adhesion andaggregation of platelets. One of the methods to evaluate “plateletactivation” in response to agonists is by measuring intracellularcalcium concentration. Another method is to quantify platelet releaseproducts in the plasma. More specifically, resting platelets maintainactive calcium efflux via a cyclic AMP activated calcium pump.Intracellular calcium concentration determines platelet activationstatus, as it is the second messenger that drives plateletconformational change and degranulation. Platelet activation beginsseconds after adhesion occurs. Thrombin is a potent platelet activator.Thrombin also promotes secondary fibrin-reinforcement of the plateletplug. Platelet activation in turn degranulates and releases factor V andfibrinogen, potentiating the coagulation cascade. Following theiractivation and F-actin polymerization, platelets must spread over intactblood vessels in the process of clot formation. Adhesion of platelets tofibrinogen is a key process in platelet aggregation, mediated byintegrins, such as αIIbβ3. WASp is important for αIIbβ3-mediated-celladhesion of platelets and megakaryocytes. Platelets contain densegranules, lambda granules and alpha granules. Activated plateletssecrete the contents of these granules through their canalicular systemsto the exterior.

In yet some further embodiments, the SMCs of the invention may decreasedegradation of WASp in any non-erythroid hematopoietic cell, forexample, any one of Granulocytes, neutrophils, Eosinophils, Basophils,Monocytes, Macrophages, and Dendritic cells (DCs).

More specifically, Granulocytes are a category of white blood cellscharacterized by the presence of granules in their cytoplasm. They arealso called polymorphonuclear leukocytes (PMN, PML, or PMNL) because ofthe varying shapes of the nucleus, which is usually lobed into threesegments. This distinguishes them from the mononuclear granulocytes.

Neutrophils are normally found in the bloodstream and are the mostabundant type of phagocyte, constituting 50% to 60% of the totalcirculating white blood cells. Once neutrophils have received theappropriate signals, it takes them about thirty minutes to leave theblood and reach the site of an infection. Neutrophils do not return tothe blood; they turn into pus cells and die. Mature neutrophils aresmaller than monocytes, and have a segmented nucleus with severalsections (two to five segments); each section is connected by chromatinfilaments. Neutrophils do not normally exit the bone marrow untilmaturity, but during infection neutrophil precursors called myelocytesand promyelocytes are released.

Neutrophils display three strategies for directly attackingmicro-organisms: phagocytosis (ingestion), release of solubleanti-microbials (including granule proteins), and generation ofneutrophil extracellular traps (NETs). The intracellular granules of thehuman neutrophil have long been recognized for their protein-destroyingand bactericidal properties.

Eosinophils play a crucial part in the killing of parasites (e.g.,enteric nematodes) because their granules contain a unique, toxic basicprotein and cationic protein (e.g., cathepsin). These cells also have alimited ability to participate in phagocytosis, but are professionalantigen-presenting cells. They are able to regulate other immune cellfunctions (e.g., CD4+ T cell, dendritic cell, B cell, mast cell,neutrophil, and basophil functions) and are involved in the destructionof tumor cells. In addition, they promote the repair of damaged tissue.

Basophils are one of the least abundant cells in bone marrow and blood.The cytoplasm of basophils contains a varied amount of granules; thesegranules are usually numerous enough to partially conceal the nucleus.Granule contents of basophils are abundant with histamine, heparin,chondroitin sulfate, peroxidase, platelet-activating factor, and othersubstances. When an infection occurs, mature basophils will be releasedfrom the bone marrow and travel to the site of infection. When basophilsare injured, they will release histamine, which contributes to theinflammatory response that helps fight invading organisms. Mast cellsalso contain many granules rich in histamine and heparin. Although bestknown for their role in allergy and anaphylaxis, mast cells play animportant protective role as well, being intimately involved in woundhealing, angiogenesis, immune tolerance, defense against pathogens, andblood-brain barrier function. The mast cell is very similar in bothappearance and function to the basophil.

Monocytes are a type of white blood cell, or leukocyte. They are thelargest type of leukocyte and can differentiate into macrophages andmyeloid lineage dendritic cells.

Macrophages are a type of white blood cell that engulfs and digestscellular debris, foreign substances, microbes, cancer cells, andanything else that does not have the types of proteins specific tohealthy body cells on its surface in a process called phagocytosis.These large phagocytes are found in essentially all tissues, where theypatrol for potential pathogens by amoeboid movement. They take variousforms (with various names) throughout the body (e.g., histiocytes,Kupffer cells, alveolar macrophages, microglia, and others), but all arepart of the mononuclear phagocyte system. Besides phagocytosis, theyplay a critical role in nonspecific defense (innate immunity) and alsohelp initiate specific defense mechanisms (adaptive immunity) byrecruiting other immune cells such as lymphocytes. For example, they areimportant as antigen presenting cells to T cells.

Dendritic cells (DCs) as used herein are antigen-presenting cells (alsoknown as accessory cells) of the mammalian immune system. Their mainfunction is to process antigen material and present it on the cellsurface to the T cells of the immune system. They act as messengersbetween the innate and the adaptive immune systems. Dendritic cells arepresent in the skin (where there is a specialized dendritic cell typecalled the Langerhans cell) and the inner lining of the nose, lungs,stomach and intestines. Once activated, DCs migrate to the lymph nodeswhere they interact with T cells and B cells to initiate and shape theadaptive immune response.

As noted above and exemplified by the following examples, WASp is a keyregulator of immune cell function. By promoting cytoskeletalpolymerization and reshaping, WASp is critical for immune cellstimulation, allowing a rapid and effective immune response. WASp isexclusively expressed in hematopoietic cells, and as such, may serve asa specific target for boosting the immune response. Activities supportedby WASp include but are not limited to immune cell activation,proliferation, chemotaxis, migration and cell-cell interactions. Mostimportantly, WASp is a master regulator of leukocyte effector functionsincluding, phagocytosis, cytolytic activity and cytokine transcriptionand secretion. Cells that are regulated by WASp include the lymphoidcells (T, B and Natural Killer cells), monocytes/macrophages, dendriticcells (DCs) and neutrophils as discussed above. WASp is also present inplatelets and in their precursors, the megakaryocytes. WASp expressionwas found to be crucial for both platelet production by megakaryocytes,as well as for their activation and effector function. WASp levels areregulated by two degradative pathways: proteolysis by the calpaincysteine protease, and ubiquitin-mediated proteolysis. The SMCs of theinvention were shown as effective agents for blocking WASp degradationand increasing its levels and function. Thus, in addition to the use ofthis strategy as therapeutic approach for treating the primary, innateimmuno-deficiencies such as WAS/XLT, the SMCs of the invention alsooffers a powerful approach for boosting the immune response in acquiredimmune deficiencies or other immune disorders. Increasing WASpexpression and function could be useful for increasing white blood cell(WBC) number and activity in immunopathologies such as leukopenia,neutropenia and thrombocytopenia. Remedying these hematologicaldisorders may be helpful following or during conventional cancertreatments such as chemotherapy and radiotherapy, diseases caused byviral, bacterial and parasitic pathogens, thrombocytopenia caused byblood loss or autoimmunity e.g. immune thrombocytopenic purpura (ITP).Furthermore, such a strategy could serve as supportive therapy for rapidreconstitution following bone marrow (BM) transplantation, ex vivoand/or in vivo expansion of hematopoietic cells for use in autologous orallogenic hematopoietic stem cell transplantation (HSCT), gene therapyor adoptive cell transfer, and upregulating the immune response againstcancers of non-hematopoietic origin.

Thus, in some embodiments, the SMC modulator of the invention may beused to restore, enhance and/or increase WASp levels and activity in acell of a subject suffering from an innate or acquired immune-relateddisorder or condition. Thus, the aforementioned SMCs are suitable forthe treatment, prevention an amelioration of immune-related disorder. An“Immune-related disorder” or “Immune-mediated disorder”, as used hereinencompasses any condition that is associated with the immune system of asubject, more specifically through inhibition of the immune system, orthat can be treated, prevented or ameliorated by reducing degradation ofa certain component of the immune response in a subject, such as theadaptive or innate immune response. An immune-related disorder mayinclude infectious condition (e.g., viral infections), metabolicdisorders and a proliferative disorder, specifically, cancer.

In some specific embodiments wherein the immune-related disorder orcondition may be a primary or a secondary immunodeficiency.

Thus, due to the wide range of activities of WASp, WASp-stabilizing SMCsare a promising therapeutic modality for a variety of innate andacquired immunodeficiencies caused by immunosuppressive treatments(chemo- and radiotherapy), pathogenic infections, cancer and HSCT.

More specifically, Immunodeficiency (or immune deficiency) is a state inwhich the immune system's ability to fight infectious disease and canceris compromised or entirely absent. Most cases of immunodeficiency areacquired (“secondary”) due to extrinsic factors that affect thepatient's immune system. Examples of these extrinsic factors includeviral infection, specifically, HIV, extremes of age, and environmentalfactors, such as nutrition. In the clinical setting, theimmunosuppression by some drugs, such as steroids, can be either anadverse effect or the intended purpose of the treatment. Examples ofsuch use are in organ transplant surgery as an anti-rejection measureand in patients suffering from an overactive immune system, as inautoimmune diseases. Immunodeficiency also decreases cancerimmunosurveillance, in which the immune system scans the cells and killsneoplastic ones.

Still further, Primary immunodeficiencies (PID), also termed innateimmunodeficiencies, are disorders in which part of the organism immunesystem is missing or does not function normally. To be considered aprimary immunodeficiency, the cause of the immune deficiency must not becaused by other disease, drug treatment, or environmental exposure totoxins). Most primary immune-deficiencies are genetic disorders; themajority is diagnosed in children under the age of one, although milderforms may not be recognized until adulthood. While there are over 100recognized PIDs, most are very rare. About 1 in 500 people in the UnitedStates are born with a primary immunodeficiency. Immune deficiencies canresult in persistent or recurring infections, autoinflammatorydisorders, tumors, and disorders of various organs.

There are several types of immunodeficiency that include, Humoral immunedeficiency (including B cell deficiency or dysfunction), which generallyincludes symptoms of hypogammaglobulinemia (decrease of one or moretypes of antibodies) with presentations including repeated mildrespiratory infections, and/or agammaglobulinemia (lack of all or mostantibody production) and results in frequent severe infections (mostlyfatal); T cell deficiency, often causes secondary disorders such asacquired immune deficiency syndrome (AIDS); Granulocyte deficiency,including decreased numbers of granulocytes (called as granulocytopeniaor, if absent, agranulocytosis) such as of neutrophil granulocytes(termed neutropenia); granulocyte deficiencies also include decreasedfunction of individual granulocytes, such as in chronic granulomatousdisease; Asplenia, where there is no function of the spleen; andComplement deficiency in which the function of the complement system isdeficient.

WASp was first discovered as the protein mutated in patients ofWiskott-Aldrich syndrome (WAS). WAS-mutations were found to cause therapid degradation of WASp, strongly reducing WASp expression in patientcells. WAS is characterized by severe clinical characteristics includingimmunodeficiency, recurrent infections, eczema, and susceptibility toautoimmune diseases and cancer, highlighting the importance of WASp forimmune cell function. Additionally, WAS, as well the milder WAS variant,X-linked thrombocytopenia (XLT), are characterized by severe bleedingcaused by reduced platelet number and size, and by disrupted bloodclotting, demonstrating the importance of WASp for platelet generationand function. WASp degradation in WAS and XLT was found to be linked tothe mechanism by which WASp levels and activity are regulated. Normally,when activated, WASp undergoes partial detachment from its chaperone,WASp Interacting Protein (WIP), allowing it to be ubiquitylated onlysine residues 76 and 81 by the Cbl E3 ubiquitin ligases, leading toits proteasomal degradation. In the primary immunodeficiencies, WAS/XLT,mutations on WASp disrupt its interaction with WIP, causing WASp to bereadily ubiquitylated and degraded, regardless of its activation state.

In some embodiments, the SMC modulator of the invention may be used torestore, enhance or increase WASp levels and activity in a cell of asubject suffering from a primary immunodeficiency. It should beappreciated that in certain embodiments, the SMCs of the invention maybe applicable for any of the PIDs disclosed herein. In yet some morespecific embodiments, such immunodeficiency may be a hereditary oracquired disorder associated with WASp dysfunction.

In more specific embodiments, hereditary disorder associated with WASpdysfunction may be at least one of Wiskott Aldrich Syndrome (WAS) andX-linked thrombocytopenia (XLT), or any condition or disorder associatedtherewith.

Increasing WASp levels can be of significant benefit not only forprimary immunodeficiency diseases (PIDD) caused by inherited or geneticdefects but also for secondary immunodeficiencies that occur when theimmune system is compromised due to environmental factors. Such factorsinclude but are not limited to radiotherapy as well as chemotherapy.While often used as fundamental anti-cancer treatments, these modalitiesare known to suppress immune function, leaving patients with anincreased risk of infection; indeed, infections were found to be aleading cause of patient death during cancer treatment. Neutropenia wasspecifically associated with vulnerability to life-threateninginfections following chemotherapy and radiotherapy. WASp plays key rolesin upregulating the proliferation and function of lymphocytes, and inenhancing the function of myeloid cells. Increasing myeloid cellactivity as well as lymphoid cell proliferation and activation by usinga WASp-stabilizing SMC treatment offers a promising approach forameliorating radiotherapy and chemotherapy-induced immune suppressionand to protect recovering cancer patients from opportunistic pathogens.Such a strategy is also expected to be generally useful in the treatmentof infections; increasing WASp expression and activity is likely toenhance key immune cell functions required for robust immune responses.These include chemotaxis and immune cell infiltration to inflammatorysites (by all WBCs), phagocytosis (by neutrophils,monocytes/macrophages, and dendritic cells (DCs)), and cell-mediatedcytotoxicity (by cytotoxic T lymphocytes (CTLs), or natural killer (NK)cells). Thus, this novel approach for stabilizing WASp using SMCs isexpected to improve clinical outcomes of secondary immune deficienciesby (1) increasing the proliferation and survival of all lymphoid cellpopulations (T, B and NK cells), and by (2) boosting the activation andfunction of both lymphoid and myeloid cells (monocytes, macrophages,dendritic cells).

Thus, in some embodiments, the SMC modulator/s of the invention may beused to restore, enhance or increase WASp levels and activity in a cellof a subject suffering from a secondary immunodeficiency. In morespecific embodiments, such secondary immunodeficiency may be caused byat least one of chemotherapy, radiotherapy, biological therapy, bonemarrow transplantation, gene therapy, adoptive cell transfer or anycombinations thereof.

More specifically, WASp was shown to promote lymphocyte growth andsurvival in both mice and humans. Proliferation of T or B lymphocytes inresponse to stimulation by anti-T cell antigen receptor (TCR) oranti-IgM, respectively, are impaired in WASp-deficient lymphocytes.Furthermore, similar proliferation defects were detected followinginteraction of WAS KO DCs with normal CD4+ and CD8+ T lymphocytes.Restoration of WASp expression has been shown to significantly mitigatethese defects. Still further, reduced leukocyte number is a common sideeffect of chemotherapy and radiotherapy, which exposes cancer patientsto life threatening infections. The approach of the present invention ofincreasing WASp levels and function using the SMCs of the invention hasthe potential to expedite hematopoietic rejuvenation following chemo-and radiotherapies by enhancing lymphocyte growth and survival.

Thus, in some embodiments, the SMC modulator of the invention may beused to restore, enhance or increase WASp levels and activity in a cellof a subject suffering from a secondary immunodeficiency caused by atleast one of chemotherapy, radiotherapy, biological therapy or anycombinations thereof.

Still further, WASp is a key regulator of actin cytoskeletonpolymerization and reshaping in all white blood cells, as well as inplatelets. As such, WASp is involved in multiple immune cell functionssuch as cellular activation, migration, cell to cell interaction,proliferation, and phagocytosis, all of which are actin dependentprocesses. Indeed, the restructuring of the actin cytoskeleton was foundto be a fundamental process controlling immune cell function. Theformation of actin filaments (F-actin) from actin monomers (G-actin) andthe complementary process of actin filament breakdown enable the actincytoskeleton to dynamically adapt to facilitate a variety of cellularactivities.

Actin filament formation is a highly regulated process; the spontaneousgeneration of filaments is prevented by the intrinsic instability ofactin dimers, necessitating the simultaneous association of three ormore actin subunits to establish a stable anchor from whichpolymerization can proceed. These initial polymers are called actinnuclei, and their formation is mediated by actin nucleation proteins.Arp2/3 is an actin nucleation complex that promotes actin filamentbranching from an existing filament, allowing the formation of complexactin structures. Arp2/3 activity depends on binding to proteins of thenucleation-promoting factor (NPF) family and WASp is a central NPF.

In some embodiments, the SMCs used by the invention may enhanceleukocyte migration and homing in subjects suffering from a secondaryimmunodeficiency caused by at least one of chemotherapy, radiotherapy,biological therapy or any combinations thereof.

In yet some further embodiments, the SMCs of the invention may induceand enhance migration and adhesion of neutrophils in a subject sufferingfrom a secondary immunodeficiency caused by at least one ofchemotherapy, radiotherapy, biological therapy or any combinationsthereof.

In yet some further embodiments, the SMCs of the invention may enhanceor increase recruitment of phagocytes in a subject suffering from asecondary immunodeficiency caused by at least one of chemotherapy,radiotherapy, biological therapy or any combinations thereof.

In some further embodiments, the SMCs of the invention may inducepolarization and migration of monocytes, specifically, dendritic cells(DC) in response to inflammatory chemokines in a subject suffering froma secondary immunodeficiency caused by at least one of chemotherapy,radiotherapy, biological therapy or any combinations thereof.

WASp deficiency, either in humans or mice, results in multipledysfunctions in most immune cell lineages, giving rise to combinedcellular and humoral immune deficiencies. These dysfunctions includesignificantly impaired formation of membrane structures such aslamelliopodia, fillopodia and podosomes in all myeloid and lymphoidimmune cells. Impaired assembly of these structures results indiminished growth and/or survival of hematopoietic cells, defectivephagocytosis due to the reduced formation of the actin-rich phagocyticcup, impaired polarization and migration, impaired homing to sites ofinflammation and compromised initiation of the adaptive immune responsein secondary lymphoid tissues. For example, abnormal homing of DCsprevent appropriate priming and activation of key effector cells such asNK cells, T cells and B cells, thereby diminishing the overallefficiency of the immune response.

Still further, it should be noted that podosomes play a crucial role incellular motility and invasion. These structures are localized in closeproximity with the leading edge of migrating cells, and their formationand function are dependent on WASp activity.

Thus, in some embodiments, the SMCs of the invention may enhance orincrease podosome formation and function in macrophages and DCs in asubject suffering from a secondary immunodeficiency caused by at leastone of chemotherapy, radiotherapy, biological therapy or anycombinations thereof.

More specifically, Podosomes are conical, actin-rich structures found onthe outer surface of the plasma membrane of animal cells. While usuallysituated on the periphery of the cellular membrane, these uniquestructures display a polarized pattern of distribution in migratingcells, situating at the front border between the lamellipodium andlamellum. Their primary purpose is connected to cellular motility andinvasion; therefore, they serve as both sites of attachment anddegradation along the extracellular matrix. Many different specializedcells exhibit these dynamic structures such as certain immune cells likemacrophages and dendritic cells, endothelial cells, osteoclasts,vascular smooth muscle cells and also invasive cancer cells.

In some embodiments, the SMCs of the invention may reduce degradation ofWASp, thereby enhancing formation of membrane structures such aslamelliopodia and fillopodia in all myeloid and lymphoid immune cells.The lamellipodium as used herein is a cytoskeletal protein actinprojection on the leading edge of the cell. It contains aquasi-two-dimensional actin mesh; the whole structure propels the cellacross a substrate. Within the lamellipodia are ribs of actin calledmicrospikes, which, when they spread beyond the lamellipodium frontier,are called filopodia.

In yet some further embodiments, the SMCs of the invention may enhance,increase or induce secretion of cytokines upon TCR activation in asubject suffering from a secondary immunodeficiency caused by at leastone of chemotherapy, radiotherapy, biological therapy or anycombinations thereof.

In certain embodiments, the SMCs of the invention may rescue NK cellfunction, specifically, the ability of NK cells to lyse susceptibletarget cells, in a subject suffering from a secondary immunodeficiencycaused by at least one of chemotherapy, radiotherapy, biological therapyor any combinations thereof.

In yet some further embodiments, the SMCs used by the invention mayrestore and enhance phagocytosis and the formation of phagocytic cup.Still further, Phagocytosis is the process by which a cell engulfs asolid particle to form an internal compartment known as a phagosome. Inan organism's immune system, phagocytosis is a major mechanism used toremove pathogens and cell debris. For example, when a macrophage ingestsa pathogenic microorganism, the pathogen becomes trapped in a phagosomewhich then fuses with a lysosome to form a phagolysosome. Within thephagolysosome, enzymes and toxic peroxides digest the pathogen.

In yet some further embodiments, the SMCs of the invention may enhance,increase or rescue macrophages or DC functions following chemotherapy orradiotherapy (phagocytic activity of bone marrow macrophages or DCs) ina subject suffering from a secondary immunodeficiency caused by at leastone of chemotherapy, radiotherapy, biological therapy or anycombinations thereof.

Still further, in some embodiments, the SMCs of the invention mayreverse the chemotherapy or radiotherapy damage to the B-cell follicle,as well as the marginal zone (MZ) architecture in a subject sufferingfrom a secondary immunodeficiency caused by at least one ofchemotherapy, radiotherapy, biological therapy or any combinationsthereof.

In some further embodiments, the SMCs of the invention may lead toreduced secretion of inflammatory cytokines, specifically, IFNγ and IL-2by the T cells upon restoration of Treg suppressive function in asubject suffering from a secondary immunodeficiency caused by at leastone of chemotherapy, radiotherapy, biological therapy or anycombinations thereof.

Still further, it should be noted that additional secondaryimmuno-deficiencies may result following bone marrow (BM)transplantation, gene therapy or adoptive cell transfer. The success ofhematopoietic stem cell transplantation (HSCT) is dependent on thetimely establishment of the graft and the reconstitution of the immunesystem, which is preceded by a period characterized by high risk forcomplications and infections, prior to engraftment. By enhancing immunecell proliferation, migration and function, the use of a WASpstabilizing SMCs of the invention to enhance the host immune system mayprovide crucial protection for recovering hematopoietic stem celltransplantation patients during this critical time frame.

Thus, in some further embodiments, the SMCs of the invention may induceand enhance migration and adhesion of neutrophils in a subject sufferingfrom a secondary immunodeficiency caused by bone marrow (BM)transplantation, gene therapy or adoptive cell transfer.

In some specific embodiments, SMCs of the invention may induce andenhance hematopoietic system reconstitution in a subject suffering froma secondary immunodeficiency caused by bone marrow (BM) transplantation,gene therapy or adoptive cell transfer.

Still further, in some embodiments, the SMCs of the invention may induceand enhance treatment of neutropenia in a subject suffering from asecondary immunodeficiency caused by bone marrow (BM) transplantation,gene therapy or adoptive cell transfer.

In yet some further embodiments, the SMCs of the invention may induceand enhance expansion of hematopoietic cells in culture, e.g., for bonemarrow transplant and use in ex vivo expansion of hematopoietic cells.In yet some further embodiments, the SMCs of the invention may be usefulin increasing the number of hematopoietic stem cells in a donor subjectin case of allogeneic HSCT.

Moreover, infectious diseases constitute a major risk to human health.It is estimated that the yearly death toll of infectious diseases is 15million. Moreover, pathogenic infections disrupt regular life routines,resulting in decreased productivity; influenza alone is estimated tocause, on average, the loss of 3.7 to 5.9 workdays per employee,annually. In the US alone, influenza is estimated to result in 140,000to 710,000 hospitalizations per year. Limiting the number of requiredhospitalizations and/or reducing hospitalization times can decreasehealthcare costs. Reducing the burden of infectious diseases can thus beexpected to have a significant economic potential.

Thus, in some embodiments, the SMC modulators of the invention may beused to restore, enhance and/or increase WASp levels and activity in acell of a subject suffering from immune-related disorder or condition,specifically, a pathologic condition caused by at least one pathogen.

By restoring WASp levels and activity, the SMCs of the invention improvethe ability of the immune system to deal with pathogenic challenges(viral, (e.g. CMV, EBV, influenza), or bacterial antigens [e.g.Mycobacterium tuberculosis, MTB; Streptococcus pneumoniae, pneumococcusor S. pneumoniae).]

In some further specific embodiments, the SMCs of the invention mayimprove ability to phagocytose pathogens (for pathogen clearance, andalso presenting foreign antigens to the adaptive arm of the immunesystem) of a subject suffering from a pathologic condition caused by atleast one pathogen.

In certain specific embodiments, the SMCs of the invention may induceformation of actin-based membrane invaginations, called phagocytic cupsin a subject suffering from a pathologic condition caused by at leastone pathogen.

In further embodiments, the SMCs of the invention may increase orenhance the ability of macrophages and DCs to phagocytose apoptoticcells in a subject suffering from a pathologic condition caused by atleast one pathogen.

Still further, in some embodiments, the SMCs of the invention mayenhance B cell activation against a pathogenic antigen (increasing IgMlevels) infectious diseases and related conditions, specifically, sepsisin a subject suffering from a pathologic condition caused by at leastone pathogen. It should be appreciated that pathogens described in moredetail hereinafter in connection with other aspects of the invention,are applicable in connection with the present aspect as well.

WASp was found to also affect platelets, as demonstrated by severebleeding in WAS/XLT patients caused by reduced platelet number, size andfunction. Consistent with these findings, WASp-stabilizing SMCsefficiently increased WASp expression levels and enhanced human plateletactivation responsible for their adhesion and coagulation (FIG. 4A-4B,FIG. 5C-5D and FIG. 11). Thus, a WASp-stabilizing SMC-based treatmentmay provide a potent therapeutic approach for thrombocytopenia.Specifically, in idiopathic thrombocytopenic purpura (ITP), SMCtreatment may be used to enhance platelet function as well as theproduction of new platelets by megakaryocytes in order to offsetantibody-mediated platelet destruction, improving clinical outcomes.

Thus, in some embodiments, the SMC modulators of the invention may beused to restore, enhance and/or increase WASp levels and activity in acell of a subject suffering from immune-related disorder or condition,specifically, thrombocytopenia.

In some further embodiments, the SMC modulators of the invention may beused to restore, enhance and/or increase WASp levels and activity in acell of a subject suffering from thrombocytopenia cause by bleeding,chemo- or radiotherapy, or autoimmunity (ITP).

In some specific embodiments, the SMC modulators of the invention may beused to restore, enhance and/or increase WASp levels and activity in acell of a subject suffering from ITP.

Still further, in some embodiments, the SMCs of the invention may leadto increase in platelet number (production from megakaryocytes),aggregation and activation in a subject suffering from thrombocytopenia,specifically, ITP.

In yet some further embodiments, the SMCs of the invention may enhanceF-actin content in a subject suffering from thrombocytopenia,specifically, ITP.

In certain embodiments, the SMCs of the invention may lead to increasedαIIbβ3 integrin activation in a subject suffering from thrombocytopenia,specifically, ITP.

Still further, in some embodiments, the SMCs of the invention mayenhance platelets adhesion and spreading capacity in a subject sufferingfrom thrombocytopenia, specifically, ITP.

In certain embodiments, the SMCs of the invention may reduce plateletclearance by macrophages and the premature release of platelets fromtheir precursors into the BM in a subject suffering fromthrombocytopenia, specifically, ITP.

Still further, SMC-based treatment in accordance with the invention mayalso be used to enhance anti-tumor immunity. CTLs, as well as NK cellsserve as the primary mediators of immune response against cancer cells.WASp was shown to be essential for NK-cancer cell conjugate formationand cytotoxic capacity. NK cells from WAS patients exhibit defectivecytotoxicity. Thus, WASp enhancing SMCs may increase the potency of thisresponse by improving the ability of the cells to migrate into thetumor, as well as improving activation and cytotoxic granule release.Furthermore, the tumor microenvironment induces suppression and reducedactivity of NK and T cells, through the secretion of inhibitory factorssuppressing the anti-tumor response, a phenomenon known as exhaustion.Increasing WASp, a mediator of activating immune signaling maycounteract this increased inhibitory signaling and allow a robust T celland NK cell mediated cytotoxic response.

Thus, in some further embodiments, the SMC modulators of the inventionmay be used to restore, enhance or increase WASp levels and activity ina cell of a subject suffering from an immune-related disorder orcondition that may be a cancer of a non-hematopoietic origin.

In yet some further embodiments, the SMCs of the invention may be usedas a supportive treatment for boosting the immune-system, specifically,cytotoxic lymphocytes activity in a subject suffering from a cancer of anon-hematopoietic origin.

In certain embodiments, the SMCs of the invention may enhance activationand function of cytotoxic lymphocytes, thereby enhancing tumorsurveillance in a subject suffering from a cancer of a non-hematopoieticorigin.

In some further embodiments, the SMC modulator used by the invention mayrestore or enhance WASp function and/or levels in a cell of a subjectsuffering from immune-related disorder or condition, for example, cancerof a non-hematopoietic origin. It should be understood that in someembodiments, the SMCs of the invention may be used as a supportivetreatment for boosting the immune-system, specifically, cytotoxiclymphocytes activity against the cancer.

The SMC modulators of the invention may be used in some embodiments, ina method for treating, preventing, inhibiting, reducing, eliminating,protecting or delaying the onset of a an innate or acquiredimmune-related disorder or condition in a subject in need thereof.

In some embodiments, the immune-related disorder or condition may be aprimary or a secondary immunodeficiency.

In yet some further embodiments, the subjects may suffer of a primaryimmunodeficiency that may be a hereditary or acquired disorderassociated with WASp dysfunction. In further embodiments, the subjectsmay suffer of a hereditary disorder associated with WASp dysfunction isat least one of Wiskott Aldrich Syndrome (WAS) and X-linkedthrombocytopenia (XLT), or any condition or disorder associatedtherewith.

In yet some further embodiments, the subjects may suffer of a secondaryimmunodeficiency that may be caused by at least one of chemotherapy,radiotherapy, biological therapy, bone marrow transplantation, genetherapy, adoptive cell transfer or any combinations thereof.

Still further, in some embodiments, the subjects may suffer of apathologic condition caused by at least one pathogen.

In further embodiments, the subjects may suffer from thrombocytopenia.In more particular embodiments, the thrombocytopenia may be cause bybleeding or as a result of chemo- or radiotherapy. Alternatively, suchthrombocytopenia may be an autoimmune disease, specifically, ITP.

In yet some additional embodiments, the SMC modulators of the inventionmay be used in treating subjects that may suffer of a cancer of anon-hematopoietic origin. In more specific embodiments, the SMCs of theinvention may be thus used as a supportive treatment for boosting theimmune-system, specifically, cytotoxic lymphocytes activity.

In yet a further aspect, the invention relates to a method formodulating degradation and/or stability of WASp in a cell. Morespecifically, the method may comprise in some embodiments the step ofcontacting the cell with an effective amount of at least one SMCmodulator of WASp degradation, or any vehicle, matrix, nano- ormicro-particle or any composition comprising the same. Morespecifically, the method comprises the step of contacting the cell withan effective amount of at least one of any of the SMC modulators of WASpas described herein.

In some aspects, the method of the invention may comprise the step ofcontacting the cell with an effective amount of at least one SMCmodulator of WASp having the general formula (I)

or a pharmaceutically acceptable salt, solvate, esters, hydrate,stereoisomer or physiologically functional derivative thereof, anycombination thereof, or any vehicle, matrix, nano- or micro-particle, orcomposition comprising the same, wherein

R₁ and R₂ are each independently from each other selected from H,straight or branched C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl,straight or branched C₁-C₁₂ alkoxy, a ring system containing five totwelve atoms, each optionally substituted by at least one of halide,hydroxyl, ester, ether, amide, amine, nitro, —C(═O)—O—(CH₂)_(n)—CH₃, R₅,or —NH—C(═O)—R₅, R₅ is an a ring system containing five to twelve atomsoptionally substituted by at least one halide or straight or branchedC₁-C₅ alkyl;

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to twelve membered saturated or unsaturated ring that mayoptionally include N, O, S, NH, C═N, C═O, S═O, or SO₂ and may beoptionally be substituted with at least one of straight or branchedC₁-C₅ alkyl, hydroxyl, halide and cyano;

L1 and L2 are each independently from each other to be absent or to beselected from —(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—,—(CH₂)_(n)—O—, S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n),—(CH₂)_(n)—N—C(═O)—, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—, —NH—(CH₂)_(n)—,—C(═O)—NH—(CH₂)_(n)—; —S—S—(CH₂)_(n)—; —O—(CH₂)_(n)—; —NH—(CH₂)_(n)—;C(═O)—(CH₂)_(n)—; —S—(CH₂)_(n)—; —NH—S(═O)_(n)—(CH₂)_(n)—,—CH₂—S—C(O)—NH—CH₂—, —(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—(NH)_(n)—C(═O)—, —(CH₂)_(n)—N—C(═O)— L1and L2 may be are each independently from each other optionallysubstituted with C₁-C₅ alkyl, a ring system containing five to twelveatoms substituted with C₁-C₅ alkyl

each n, is an integer being independently from each other selected frombe 0 to 5;

R₃ and R₄ are each independently from each other be absent or selectedfrom a ring system containing five to 15 atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), (O)₂, —C(O)—CH₃, —C(O)—O—CH₃, halide, CF₃, nitro, amide, or R₅, R₅is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight or branched C₁-C₅ alkyl.

The invention further provides in some aspects thereof, a method formodulating degradation of WASp in a cell. More specifically, the methodmay comprise in some embodiments the step of contacting the cell with aneffective amount of at least one SMC modulator of WASp degradation, orany vehicle, matrix, nano- or micro-particle or any compositioncomprising the same. In more specific embodiments, the SMC/s used by themethods of the invention may have the general formula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other may be absent orselected from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—, —(CH₂)—, —(CH₂)—O—,—NH—(CH₂)—, and each optionally substituted with C₁-C₅ alkyl, a ringsystem containing five to twelve atoms optionally substituted with C₁-C₅alkyl;

R₃ and R₄ are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl.

In some specific embodiments, the SMC modulator used by the methods ofthe invention may be at least one pharmaceutically acceptable salts orhydrates of the compounds of Formula XI, or in some further specificembodiments, pharmaceutically acceptable salts or hydrates of thecompounds of Formula XII. In more specific and non-limiting example,such compounds may include:

1-(2,6-Dimethyl-piperidin-1-yl)-2-[5-(quinolin-8-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-ethanone(designated herein as SMC 34); or

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-3-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(designated herein as SMC 34.7).

In yet some further particular embodiments, specific and non-limitingexamples of the SMC modulators that may be used by the methods of theinvention, may be pharmaceutically acceptable salts or hydrates of thecompounds of Formula XI, or in some further specific embodiments,pharmaceutically acceptable salts or hydrates of the compounds ofFormula XII, that may include:

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-m-tolyloxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.1);

2-(5-Methyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-piperidin-1-yl-ethanone(SMC 34.3);

N-Cyclohexyl-2-[5-(naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetamide(SMC 34.4);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.5);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-pyridin-3-yl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.6);

2-(5-Phenoxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-pyrrolidin-1-yl-ethanone(34.8 SMC);

2-(Quinolin-8-yloxymethyl)-oxazole-4-carboxylic acid(tetrahydro-pyran-2-ylmethyl)-amide (SMC 34.10)

2-[4-Phenyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-1-piperidin-1-yl-ethanone(SMC 34.11);

1-Piperidin-1-yl-2-(5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.12);

2-[4-Methyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide(SMC 34.13).

In yet some further particular embodiments, an additional example of aSMC modulator that may be used by the method of the invention, may bethe compound:

[5-(Naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetic acidmethyl ester (SMC 34.9).

In yet some further alternative and particular embodiments, the SMCmodulator used by the method invention may be any compound defined byFormula XI, Formula XII, with the proviso that the compound is not anyof the compounds detailed below:

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-m-tolyloxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.1).

2-(5-Methyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-piperidin-1-yl-ethanone(SMC 34.3)

N-Cyclohexyl-2-[5-(naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetamide(SMC 34.4);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanoneSMC 34.5);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-pyridin-3-yl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.6);

2-(5-Phenoxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-pyrrolidin-1-yl-ethanone(SMC 34.8)

2-(Quinolin-8-yloxymethyl)-oxazole-4-carboxylic acid(tetrahydro-pyran-2-ylmethyl)-amide (SMC 34.10).

2-[4-Phenyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-1-piperidin-1-yl-ethanone(SMC 34.11).

1-Piperidin-1-yl-2-(5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.12).

2-[4-Methyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide(SMC 34.13).

In yet some further particular embodiments, the SMC modulator that maybe used by the method of the invention, may be any of the compoundsdisclosed by the invention provided that said compound is not thecompound:

[5-(Naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetic acidmethyl ester (SMC 34.9).

In yet some further particular embodiments, the SMC modulator that maybe used by the method of the invention, may be any of the compoundsdisclosed by the invention provided that said compound is not thecompound:

8-(5-Isopropyl-[1,3,4]oxadiazol-2-ylmethoxy)-quinoline (34.2).

In yet some further specific embodiments, the SMC modulator used by themethods of the invention may be at least one pharmaceutically acceptablesalts or hydrates of the compounds of Formula XI, or in some furtherspecific embodiments, pharmaceutically acceptable salts or hydrates ofthe compounds of Formula XIII. In more specific and non-limitingexample, such compounds may include:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide(Designated herein as SMC#33); or

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester

(Designated herein as SMC#30).

In yet some further embodiments, the method comprises the step ofcontacting the cell with an effective amount ofN′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamidehaving a structure:

(denoted herein as SMC #33)

In some other embodiments, the method comprises the step of contactingthe cell with an effective amount of1-(2,6-dimethylpiperidin-1-yl)-2-[[5-(quinolin-8-yloxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]ethanonehaving a structure:

(denoted herein as SMC #34)

In some embodiments, the method comprises the step of contacting thecell with an effective amount of ethyl 4-[[3-(4-acetylpiperazin-1-yl)sulfonylbenzoyl]amino]benzoate having a structure:

(denoted herein as SMC #23)

In some embodiments, the method comprises the step of contacting thecell with an effective amount of10-(3-Chloro-benzyl)-8-(4-methyl-piperazine-1-carbonyl)-5,5-dioxo-5,10-dihydro-5λ6-dibenzo[b,f][1,4]thiazepin-11-onehaving a structure

(denoted herein as SMC #26)

In accordance with some other aspects, the method comprises the step ofcontacting the cell with an effective amount of at least one SMCmodulator of WASp having the general formula (V):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

each one of X, Y, Z, V, W, T and S may be selected from N, NH and C,

R₆ and R₇ are the same or are different and are independently selectedfrom each other may be L3-R₈,

L3 may be selected from —(CH₂)_(n), —NH—C(O) and C(O)—NH, S(O)₂, C(O), nis an integer between 0 to 5;

R₈ may be selected from a ring system containing five to twelve atoms,each optionally substituted by at least one of straight or branchedC₁-C₅ alkyl, halide, hydroxyl, ester, ether, amide, nitro and hydroxyl,CF₃.

In some embodiments, the method comprises the step of contacting thecell with an effective amount of

3-[(3-fluorophenyl)methyl]-5-[1-[2-(trifluoromethyl)phenyl]sulfonylpiperidin-4-yl]-2H-triazolo[4,5-d]pyrimidin-7-one

(Designated herein as SMC #32).

In accordance with some other aspects, the method comprises the step ofcontacting the cell with an effective amount of at least one SMCmodulator of WASp having the general formula (IX):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

L4 may be absent or may be selected from —(CH₂)_(n)—, —S—(CH₂)_(n)—,—(CH₂)_(n)—S—; n is an integer between 0 and 5;

R₁₀ and R11 are each independently from each other absent or selectedfrom a ring system containing five to twelve atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), —C(O)—CH₃, —C(O)—O—CH₃, halide, nitro, NH₂.

In some embodiments, L4 is selected from —S—(CH₂)—. In some otherembodiments, R₁₀ and R11 are each independently from each other absentor selected from triazine, piperidine each optionally substituted withat least one NH₂.

In accordance with some other aspects, the method comprises the step ofcontacting the cell with an effective amount of at least one SMCmodulator of WASp having the general formula (X):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁₃ and R14 are each independently from each other absent or selectedfrom a ring system containing five to twelve atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, C₁-C₅alkoxy, (═O), (═S), —C(O)—CH₃, —C(O)—O—CH₃, halide, nitro, NH₂,NH—C(O)—CH₃.

In some other embodiments, R₁₀ and R11 are each independently from eachother absent or selected from aryl each optionally substituted with atleast one OCH, NH—C(O)—CH₃.

Still further, in some embodiments, the compound used in the methods ofthe invention may be the SMC#6, specifically,N-[(2R,4R,6S)-2-(4-chlorophenyl)-6-(1-methylbenzotriazol-5-yl)oxan-4-yl]acetamide.

In yet some alternative embodiments, the invention provides methodsusing of any of the SMCs disclosed herein, provided that said SMC is notSMC#6, specifically,N-[(2R,4R,6S)-2-(4-chlorophenyl)-6-(1-methylbenzotriazol-5-yl)oxan-4-yl]acetamide.

In more specific embodiments, the method of the invention may use any ofthe SMC modulator/s of the invention as defined herein, or anycombinations thereof.

In some specific embodiments, the modulation of WAPs degradation by themethods of the invention may result in reduced WASp degradation in acell.

In more specific embodiments, the methods of the invention may lead toreduced degradation of WASp and thereby restores, enhances and/orextends at least one of WASp levels and function in the treated cell.

In some specific embodiments, the cell modulated by the method of theinvention may be a hematopoietic cell, more specifically, any cell ofthe lymphoid or the myeloid lineages. In yet some further embodiments,the hematopoietic cell according to the invention may be a non-erythroidhematopoietic cell.

In yet some further embodiments, the SMCs used by the methods of theinvention may modulate WASp levels and/or function in a non-erythroidhematopoietic cell that may be least one of a lymphocyte, platelet,megakaryocyte, granulocyte and monocyte.

In some embodiments, the WASp function in a cell restored, extended orenhanced by the SMCs used by the methods of the invention may be atleast one of cell activation, cell proliferation, cell migration, cellhoming, cell spreading, cell aggregation, elevation in intracellularcalcium concentration, cell adhesion, phagocytosis, and cytolyticactivity.

In yet some further embodiments, the SMCs used by the methods of theinvention may modulate, specifically, enhance and/or restore WASp levelsand/or function as described above, in a cell of a subject sufferingfrom an innate or acquired immune-related disorder or condition.

In some embodiments, the immune-related disorder or condition may be aprimary or a secondary immunodeficiency. In yet some further specificembodiments, the primary immunodeficiency may be a hereditary oracquired disorder associated with WASp dysfunction. Still further, insome embodiments, the hereditary disorder associated with WASpdysfunction may be at least one of WAS and XLT, or any condition ordisorder associated therewith.

In yet some further embodiments, acquired disorders or conditionsassociated with WAS and XLT, may include autoimmune disorders,inflammatory disorders, atopic eczema.

As shown by the EXAMPLES, application of the WASp-binding SMC modulatorsof the invention that potentially protect WASp from degradation, lead toincreased WASp expression in T-cell lines, primary lymphocytes,megakaryocytes and platelets by reducing WASp ubiquitylation (FIGS. 4,6, 7, 12, 14 and 15). This increase in WASp levels by the SMC modulatorsenhanced WASp-dependent cellular functions, including cellularactivation, intra-cellular calcium influx, proliferation and migration(FIG. 8, 9-11). In addition, treatment with SMCs not only reconstitutedthe expression of common WASp mutants but also restored the function ofmutant WASp-expressing cells. (FIG. 14, 15).

As therapeutic agents, SMCs have several advantages over proteins andpeptides, including superior bio-availability and less immunogenicity.Currently MSCs cannot be tested in vivo due to the lack of valid animalmodels. The only available animal model for studying WAS is the WASknockout mouse, which is completely devoid of the WAS gene and as suchis not a suitable model for testing post translational pathways. Becausethe rarity of WAS/XLT, the inventors established WASp-deficient T celllines, reconstituted with WAS/XLT mutant WASp, as a mid-step towardsevaluation of WASp-protecting SMCs. The data presented in EXAMPLES 4, 5and 6 provide proof of concept for using rationally-selectedWASp-binding SMCs as a suitable treatment for WASp dysfunctiondisorders.

Therefore, attenuation of WASp degradation by WASp-binding SMCmodulators of the invention is suggested as a promising treatment optionfor Wiskott-Aldrich Syndrome and X-linked thrombocytopenia.

In yet some alternative embodiments, the secondary immunodeficiency maybe caused by at least one of chemotherapy, radiotherapy, biologicaltherapy, bone marrow transplantation, gene therapy, adoptive celltransfer or any combinations thereof.

It should be appreciated that in case of bone marrow transplantations,by restoring, enhancing and/or extending at least one of WASp levels andfunction in a cell using the SMC modulators described herein, theinvention further encompasses an in vitro method for expansion of cellsin culture, e.g., for bone marrow transplant and use in ex vivoexpansion of hematopoietic cells.

In yet some further embodiments, the method of the invention may beapplicable in restoring, enhancing or extending at least one of WASplevels and function in a cell of a subject suffering from a pathologiccondition caused by at least one pathogen.

In yet some further embodiments, the method of the invention may beapplicable in restoring, enhancing or extending at least one of WASplevels and function in a cell of a subject suffering fromthrombocytopenia. More specifically, thrombocytopenia cause by bleeding,chemo- or radiotherapy, or autoimmunity (ITP).

In yet some certain embodiments, the method of the invention may beapplicable in restoring, enhancing and/or extending at least one of WASplevels and function in a cell of a subject suffering from cancer of anon-hematopoietic origin. In more specific embodiments, such method maybe used as a supportive treatment for boosting the immune-system,specifically, cytotoxic lymphocytes activity in the treated subjects.

Still further, in some embodiments, for modulating the WASplevels/activity in a cell, the SMC modulators of the invention may becontacted with the cell. The term “contacting” as used herein, means tobring, put, incubate or mix together. As such, a first item is contactedwith a second item when the two items are brought or put together, e.g.,by touching them to each other or combining them. In the context of thepresent invention, the term “contacting” includes all measures or steps,which allow interaction between the compounds of the invention and thecells or subjects to be modulated, as specified herein after.

As indicated above, the present invention provides methods formodulating WASp degradation levels in a cell. The term modulation asused herein refers to reduction or alternatively, to elevation of WASpdegradation in said cell.

In yet more specific embodiments, the SMC of the invention may lead todecrease, reduction, elimination, attenuation or inhibition of the WASpdegradation of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or about 100% in a cell contacted with, or in asubject administered with the SMC of the invention as compared with acell or a subject not treated with the SMC of the invention.

In yet a further aspect, the invention relates to a method for treating,preventing, inhibiting, reducing, eliminating, protecting or delayingthe onset of an innate or acquired immune-related disorder or conditionin a subject in need thereof. More specifically, the method may compriseadministering to the subject a therapeutically effective amount of atleast one SMC modulator of WASp as described herein.

In accordance with some aspects, the method for treating, preventing,inhibiting, reducing, eliminating, protecting or delaying the onset ofan innate or acquired immune-related disorder or condition in a subjectin need comprise administering to the subject a therapeuticallyeffective amount of at least one SMC modulator of WASp having thegeneral formula (I)

or a pharmaceutically acceptable salt, solvate, esters, hydrate,stereoisomer or physiologically functional derivative thereof, anycombinations thereof, or of any vehicle, matrix, nano- or micro-particleor composition comprising the same, wherein R₁ and R₂ are eachindependently from each other selected from H, straight or branchedC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl, straight or branched C₁-C₁₂alkoxy, a ring system containing five to twelve atoms, each optionallysubstituted by at least one of halide, hydroxyl, ester, ether, amide,amine, nitro, —C(═O)—O—(CH₂)_(n)—CH₃, R₅, or —NH—C(═O)—R₅, R₅ is an aring system containing five to twelve atoms optionally substituted by atleast one halide or straight or branched C₁-C₅ alkyl;

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to twelve membered saturated or unsaturated ring that mayoptionally include N, O, S, NH, C═N, C═O, S═O or SO₂ and may beoptionally be substituted with at least one of straight or branchedC₁-C₅ alkyl, hydroxyl, halide and cyano;

L1 and L2 are each independently from each other selected to be absentor from —(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—,—(CH₂)_(n)—O—, S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n),—(CH₂)_(n)—N—C(═O)—, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—, —NH—(CH₂)_(n)—,—C(═O)—NH—(CH₂)_(n)—; —S—S—(CH₂)_(n)—; —O—(CH₂)_(n)—; —NH—(CH₂)_(n)—;C(═O)—(CH₂)_(n)—; —S—(CH₂)_(n)—; —NH—S(═O)_(n)—(CH₂)_(n)—,—CH₂—S—C(O)—NH—CH₂—, —(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—(NH)_(n)—C(═O)—, —(CH₂)_(n)—N—C(═O)— L1and L2 may be are each independently from each other optionallysubstituted with C₁-C₅ alkyl, a ring system containing five to twelveatoms substituted with C₁-C₅ alkyl

each n, is an integer being independently from each other selected frombe 0 to 5;

R₃ and R₄ are each independently from each other be absent or selectedfrom a ring system containing five to 15 atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), (O)₂, —C(O)—CH₃, —C(O)—O—CH₃, halide, CF₃, nitro, amide, or R₅, R₅i an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight or branched C₁-C₅ alkyl.

In some specific embodiments, the method of the invention may use any ofthe SMC modulator/s defined by the invention or any combinationsthereof.

In some embodiments of this aspect, the SMC isN′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamidehaving a structure:

In some embodiments of this aspect, the SMC is1-(2,6-dimethylpiperidin-1-yl)-2-[[5-(quinolin-8-yloxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]ethanonehaving a structure

In some embodiments of this aspect, the SMC is ethyl4-[[3-(4-acetylpiperazin-1-yl)sulfonylbenzoyl]amino]benzoate having astructure

In some embodiments of this aspect, the SMC is10-(3-Chloro-benzyl)-8-(4-methyl-piperazine-1-carbonyl)-5,5-dioxo-5,10-dihydro-5λ6-dibenzo[b,f][1,4]thiazepin-11-one having a structure

In still some further aspects thereof, the invention provides a methodfor treating, preventing, inhibiting, reducing, eliminating, protectingor delaying the onset of an innate or acquired immune-related disorderor condition in a subject in need thereof. More specifically, the methodmay comprise administering to the treated subject a therapeuticallyeffective amount of at least one SMC modulator of WASp having thegeneral formula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other may be absent orselected from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—, —(CH₂)—, —(CH₂)—O—,—NH—(CH₂)—, and each optionally substituted with C₁-C₅ alkyl, a ringsystem containing five to twelve atoms optionally substituted with C₁-C₅alkyl;

R₃ and R₄ are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl.

In some particular embodiments, specific and non-limiting examples ofthe SMC modulators used by the methods of the invention, may bepharmaceutically acceptable salts or hydrates of the compounds ofFormula XI, or in some further specific embodiments, pharmaceuticallyacceptable salts or hydrates of the compounds of Formula XII, that mayinclude:

1-(2,6-Dimethyl-piperidin-1-yl)-2-[5-(quinolin-8-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-ethanone(designated herein as SMC 34); or

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-3-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(designated herein as SMC 34.7).

In yet some further particular embodiments, specific and non-limitingexamples of the SMC modulators that may be used by the methods of theinvention, may be pharmaceutically acceptable salts or hydrates of thecompounds of Formula XI, or in some further specific embodiments,pharmaceutically acceptable salts or hydrates of the compounds ofFormula XII, that may include:

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-m-tolyloxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.1);

2-(5-Methyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-piperidin-1-yl-ethanone(SMC 34.3);

N-Cyclohexyl-2-[5-(naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetamide(SMC 34.4);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.5);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-pyridin-3-yl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.6);

2-(5-Phenoxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-pyrrolidin-1-yl-ethanone(SMC 34.8);

2-(Quinolin-8-yloxymethyl)-oxazole-4-carboxylic acid(tetrahydro-pyran-2-ylmethyl)-amide (SMC 34.10);

2-[4-Phenyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-1-piperidin-1-yl-ethanone(SMC 34.11);

1-Piperidin-1-yl-2-(5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.12);

2-[4-Methyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide(SMC 34.13);

In yet some further particular embodiments, an additional example of aSMC modulator that may be used by the method of the invention, may bethe compound:

[5-(Naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetic acidmethyl ester (SMC 34.9).

In yet some further particular embodiments, an additional example of aSMC modulator that may be used by the method of the invention, may bethe compound:

In yet some further particular embodiments, an additional example of aSMC modulator that may be used by the method of the invention, may bethe compound:

8-(5-Isopropyl-[1,3,4]oxadiazol-2-ylmethoxy)-quinoline (34.2).

In yet some further alternative and particular embodiments, the SMCmodulator used by the method invention may be any compound defined byFormula XI, Formula XII, with the proviso that the compound is not anyof the compounds detailed below:

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-m-tolyloxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.1);

2-(5-Methyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-piperidin-1-yl-ethanone(SMC 34.3);

N-Cyclohexyl-2-[5-(naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetamide(SMC 34.4);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.5);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-pyridin-3-yl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.6);

2-(5-Phenoxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-pyrrolidin-1-yl-ethanone(SMC 34.8);

2-(Quinolin-8-yloxymethyl)-oxazole-4-carboxylic acid(tetrahydro-pyran-2-ylmethyl)-amide (SMC 34.10);

2-[4-Phenyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-1-piperidin-1-yl-ethanone(SMC 34.11);

1-Piperidin-1-yl-2-(5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.12);

2-[4-Methyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide(SMC 34.13).

In yet some further particular embodiments, the SMC modulator that maybe used by the method of the invention, may be any of the compoundsdisclosed by the invention provided that said compound is not thecompound:

[5-(Naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetic acidmethyl ester (SMC 34.9).

In yet some further particular embodiments, the SMC modulator that maybe used by the method of the invention, may be any of the compoundsdisclosed by the invention provided that said compound is not thecompound:

8-(5-Isopropyl-[1,3,4]oxadiazol-2-ylmethoxy)-quinoline (34.2).

In some particular embodiments, specific and non-limiting examples ofthe SMC modulators used by the methods of the invention, may bepharmaceutically acceptable salts or hydrates of the compounds ofFormula XI, or in some further specific embodiments, pharmaceuticallyacceptable salts or hydrates of the compounds of Formula XIII, that mayinclude:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide

(Designated herein as SMC#33); or

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester

(Designated herein as SMC#30).

In accordance with some other aspects, the method for treating,preventing, inhibiting, reducing, eliminating, protecting or delayingthe onset of an innate or acquired immune-related disorder or conditionin a subject in need comprise administering to the subject atherapeutically effective amount of at least one SMC modulator of WAShaving the general formula (V):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

each one of X, Y, Z, V, W, T and S may be selected from N, NH and C,

R₆ and R₇ are the same or are different and are independently selectedfrom each other may be L3-R₈,

L3 may be selected from —(CH₂)_(n), —NH—C(O) and C(O)—NH, S(O)₂, C(O),

n is an integer between 0 to 5;

R₈ may be selected from a ring system containing five to twelve atoms,each optionally substituted by at least one of straight or branchedC₁-C₅ alkyl, halide, hydroxyl, ester, ether, amide, nitro and hydroxyl,CF₃.

In some embodiments of this aspect, the method comprises administeringto the subject a therapeutically effective amount of

3-[(3-fluorophenyl)methyl]-5-[1-[2-(trifluoromethyl)phenyl]sulfonylpiperidin-4-yl]-2H-triazolo[4,5-d]pyrimidin-7-one

(Designated herein as SMC #32).

In accordance with some other aspects, the method for treating,preventing, inhibiting, reducing, eliminating, protecting or delayingthe onset of an innate or acquired immune-related disorder or conditionin a subject in need comprise administering to the subject atherapeutically effective amount of at least one SMC modulator of WASphaving the general formula (IX):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

L4 may be absent or may be selected from —(CH₂)_(n)—, —S—(CH₂)_(n)—,—(CH₂)_(n)—S—;

n is an integer between 0 and 5;

R₁₀ and R₁₁ are each independently from each other absent or selectedfrom a ring system containing five to twelve atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), —C(O)—CH₃, —C(O)—O—CH₃, halide, nitro, NH₂.

In some embodiments, L4 is selected from —S—(CH₂)—. In some otherembodiments, R₁₀ and R11 are each independently from each other absentor selected from triazine, piperidine each optionally substituted withat least one NH₂.

In accordance with some other aspects, the method for treating,preventing, inhibiting, reducing, eliminating, protecting or delayingthe onset of an innate or acquired immune-related disorder or conditionin a subject in need comprise administering to the subject atherapeutically effective amount of at least one SMC modulator of WASphaving the general formula (X):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁₃ and R₁₄ are each independently from each other absent or selectedfrom a ring system containing five to twelve atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, C₁-C₅alkoxy, (═O), (═S), —C(O)—CH₃, —C(O)—O—CH₃, halide, nitro, NH₂,NH—C(O)—CH₃.

In some other embodiments, R₁₀ and R11 are each independently from eachother absent or selected from aryl each optionally substituted with atleast one OCH, NH—C(O)—CH₃.

Still further, in some embodiments, the compound used in the methods ofthe invention may be the SMC#6, specifically,N-[(2R,4R,6S)-2-(4-chlorophenyl)-6-(1-methylbenzotriazol-5-yl)oxan-4-yl]acetamide.

In yet some alternative embodiments, the invention provides methodsusing of any of the SMCs disclosed herein, provided that said SMC is notSMC#6, specifically,N-[(2R,4R,6S)-2-(4-chlorophenyl)-6-(1-methylbenzotriazol-5-yl)oxan-4-yl]acetamide.

In some embodiments, the methods of the invention may be particularlyapplicable for treating an immune-related disorder or condition that maybe a primary or a secondary immunodeficiency. Specifically, any of theprimary or a secondary immunodeficiency disorders described hereinbefore in connection with other aspects of the invention.

In yet some further specific embodiments, the methods of the inventionmay be applicable in the treatment of a primary immunodeficiency,specifically, a hereditary or acquired disorder associated with WASpdysfunction.

In more specific embodiments, such hereditary disorder associated withWASp dysfunction may be at least one of WAS and XLT, or any condition ordisorder associated therewith.

In some specific embodiments the SMC modulators of the invention may besuitable for treating Wiskott-Aldrich Syndrome.

The “Wiskott-Aldrich syndrome” (WAS) is a rare (affects 4 of every 1million males worldwide) X-linked disorder characterized bythrombocytopenia, small platelets, eczema, recurrent infections,immunodeficiency, and a high incidence of autoimmune diseases andmalignancies. A classic WAS phenotype is generally associated withnull-mutations of the WAS gene located on the short arm of the Xchromosome (Xp11.22) that encode the WAS protein (WASp).

It should be appreciated that in certain embodiments, the SMCs of theinvention may be relevant for the treatment of any disease caused by amutation in the WAS gene. In yet some further embodiments, the SMCs ofthe invention may be applicable for any WASp mutant, wherein thetranslated WAS protein maintains or retains, at least in part, theWASp-homology-1 (WH1) domain corresponding to the interaction site withWIP also named WASp degradation pocket.

The term “mutation” is used herein to describe any inherited or sporadicchange in the nucleotide sequence or arrangement of DNA that results ina dysfunctional or absent protein including, but not limited to thefollowing: nucleotide substitutions (e.g. missense mutations, nonsensemutations, non-stop mutations, RNA processing mutations, splice-sitemutations, regulatory mutations, nucleotide transitions and nucleotidetransversions), insertions or deletions of one or more nucleotides,truncations, duplications of any nucleotide sequence, repeat expansionmutations (e.g. trinucleotide repeats, etc.) and frameshift mutations.In some embodiments, the SMCs of the invention are particularly relevantfor reducing degradation of WASp with a missense mutation. In otherembodiments, the SMCs of the invention are particularly relevant forreducing degradation of WASp with any mutation as discussed above. Inyet some further embodiments, the SMCs of the invention may beapplicable for any known WASp mutation, specifically any of the WASpmutations disclosed herein after or any combinations thereof. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of WASp R86C mutant (specifically,substitution of Arginine to Cysteine in position 86 of WASp as denotedby the amino acid sequence of SEQ ID NO. 1). In other specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of WASp Y107C mutant (specifically, substitution ofTyrosine to Cysteine in position 107 of WASp as denoted by the aminoacid sequence of SEQ ID NO. 1). In some specific embodiments, the SMCsof the invention are particularly relevant for reducing degradation ofWASp A134T mutant (specifically, substitution of Alanine to Threonine inposition 134 of WASp as denoted by the amino acid sequence of SEQ ID NO.1). In some specific embodiments, the SMCs of the invention areparticularly relevant for reducing degradation of WASp T45M mutant. Insome specific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of WASp R86S mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of WASp R86G mutant. In some specific embodiments,the SMCs of the invention are particularly relevant for reducingdegradation of WASp R86H mutant. In some specific embodiments, the SMCsof the invention are particularly relevant for reducing degradation ofWASp R86L mutant. In some specific embodiments, the SMCs of theinvention are particularly relevant for reducing degradation of WASpR211stop codon mutant (truncation). In some specific embodiments, theSMCs of the invention are particularly relevant for reducing degradationof WASp mutant with deletion of Exon 8. In some specific embodiments,the SMCs of the invention are particularly relevant for reducingdegradation of WASp E31K mutant. In some specific embodiments, the SMCsof the invention are particularly relevant for reducing degradation ofWASp E133K mutant. In some specific embodiments, the SMCs of theinvention are particularly relevant for reducing degradation of WASpE133D mutant. In some specific embodiments, the SMCs of the inventionare particularly relevant for reducing degradation of WASp C73Y mutant.In some specific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of WASp C73W mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of WASp V75M mutant. In some specific embodiments,the SMCs of the invention are particularly relevant for reducingdegradation of WASp V51F mutant. In some specific embodiments, the SMCsof the invention are particularly relevant for reducing degradation ofWASp W97C mutant. In some specific embodiments, the SMCs of theinvention are particularly relevant for reducing degradation of WASpC43W mutant. In some specific embodiments, the SMCs of the invention areparticularly relevant for reducing degradation of WASp A56V mutant. Insome specific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of WASp P58A mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp S24P mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp S24F mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp L27F mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp L35H mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp L39P mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp A47D mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp T48I mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp P58R mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp W64R mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp F74S mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp K76T mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp D77H mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp D77G mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp S82P mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp W97X mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp Q99X mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp E100D mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp L105P mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp G119E mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp C122X mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp G125R mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp L126P mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp F128L mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp F128S mutant. In some specificembodiments, the SMCs of the invention are particularly relevant forreducing degradation of the WASp E131K, E133K double mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp E131K, R86K double mutant.In some specific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp A134V mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp S228X mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp A236G mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp E285X mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp Q297X mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp M307V mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp R321X mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp P361T mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp R364X mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp I481N mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp X503S mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp V75L mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp V75L mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp S2T mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp M6I mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp L39P mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp D58A mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp E67K mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp Q80R mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp Y83X mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp Y88C mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp G89D mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp G119R mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp N169X mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp H180N mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp S242C mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp G334X mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp G363X mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp G477K mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp D485N mutant. In somespecific embodiments, the SMCs of the invention are particularlyrelevant for reducing degradation of the WASp D485G mutant.

It should be appreciated that the specific WASp mutants referred toherein are indicated using the conventional nomenclature for one lettercode of the substituted amino acid residue. Specifically, Ala or A foralanine, Arg or R for arginine, Asn or N for Asparagine, Asp or D forAspartic acid, Cys or C for Cysteine, Glu or E for Glutamic acid, Gln orQ for Glutamine, Gly or G for Glycine, His or H for Histidine, Ile or Ifor Isoleucine, Leu or L for Leucine, Lys or K for Lysine, Met or M forMethionine, Phe or F for Phenylalanine, Pro or P for Proline, Ser or Sfor Serine, Thr or T for Threonine, Trp or W for Tryptophan, Tyr or Yfor Tyrosine and Val or V for Valine. Still further, Asx or B foraspartic acid or aspargine, Llx or Z for Glutamine or Glutamic acid, Xleor J for Leucine or Isoleucine, and Xaa or X, for any amino acid. Thesubstituted position is referred to herein in relation to the wild typesequence of the human WASp as denoted by the amino acid sequence of SEQID NO. 1.

In some specific embodiments, SMC#34, is particularly relevant forreducing degradation of the WASp R86C mutant or the WASp Y107C mutant orthe WASp A134T mutant.

In further specific embodiments, SMC#6 is particularly relevant forreducing degradation of the WASp R86C mutant or the WASp Y107C mutant orthe WASp A134T mutant. In other specific embodiments, SMC#30 isparticularly relevant for reducing degradation of the WASp R86C mutantor the WASp Y107C mutant or the WASp A134T mutant.

In more specific embodiments, SMC#33 is particularly relevant forreducing degradation of the WASp R86C mutant or the WASp Y107C mutant orthe WASp A134T mutant.

It should be appreciated that the method of the invention may beapplicable also to disorders or conditions associated with WAS. Morespecifically, the intrinsic defects in phagocytosis, chemotaxis,cytotoxicity, apoptosis and cell signaling appear to contribute to morethan just the immunodeficiency observed in WAS that has high incidenceof autoimmunity, inflammatory conditions and vasculitis. Still further,WAS results from a hematopoietic-cell-specific defect in actinreorganization and cell signaling due to WASp deficiency. Patientslacking WASp expression or expressing abnormal WASp have NK cells withdecreased cytolytic capacity. Clinically, patients with WAS aresusceptible to herpes viruses and can develop HLH.

Clinically, the genetic defect produces cellular and humoralimmunodeficiency, high susceptibility to autoimmune diseases, andincreased risk for developing hematologic malignancies. This is theclassic presentation, but the disease has a broad clinical spectrum thattends to correlate with the location and nature of the causativemutation. More than 300 mutations have been described, and the clinicalpicture varies from mild microthrombocytopenia alone (also calledX-linked thrombocytopenia) to the full-blown syndrome.

Still further, in other specific embodiments the SMC modulators of theinvention may be suitable for treating X-linked thrombocytopenia.

More specifically, a milder phenotype, “X-linked thrombocytopenia”(XLT), is often associated with mutations that result in expression ofmutated protein. XLT patients have either no or very mild eczema and fewproblems, if any, with infections, autoimmunity, and malignancy. Aminoacid substitutions within the guanosine triphosphatase-binding domain ofWASP interfere with the intramolecular autoinhibitory mechanism,resulting in gain of function impaired actin polymerization, causingX-linked neutropenia. By definition, WAS and XLT patients havecongenital thrombocytopenia in the range of 20,000 to 60,000/L andmicroplatelets, but normal numbers of megakaryocytes. Hemorrhagicproblems may be mild, consisting of bruises and petechiae, or serious,including gastrointestinal and central nervous system hemorrhages.Patients with classic WAS acquire bacterial, fungal, and viralinfections.

Because of the poor long-term outcome, early allogeneic hematopoieticstem cell transplantation is the treatment of choice, however, thisprocedure is associated with many risks. Haploidentical transplantationis not recommended. Splenectomy ameliorates the bleeding tendency byincreasing the number of blood platelets. However, splenectomysubstantially increases the risk of septicemia, sometimes in spite ofantibiotic prophylaxis, and often results in fatal bacterial infections.Patients with XLT have better prognosis, but may develop complicationsincluding serious bleeding, autoimmune diseases, and malignancies.Furthermore patients that are treated by splenectomy are not chosen forbone marrow transplantation or gene therapy due to their impaired immuneresponse.

Still further, as indicated above, it should be appreciated that theinvention further encompasses methods for treating WAS, XLT and anyconditions or disorders associated therewith. Such disorders orconditions may include but are not limited to Microthrombocytopenia,inflammatory skin chronic conditions such as eczema, autoimmuneconditions, immunodeficiency and tumors.

The term “Microthrombocytopenia” as used herein, refers to a decrease inthrombocyte (platelet) number and to a small size of generatingplatelets, which is the most consistent feature of WASP-associateddisease. The mechanism of the abnormal size remains incompletelyunderstood. Mutant WASP is uniformly absent in platelets, even in themildest patient phenotypes, suggesting a direct role of WASP deficiencyin producing thrombocytopenia (a decrease in platelet number) andmicrothrombocytopenia.

Among clinical manifestations, hemorrhages are frequent (80% incidence)in WAS and XLT patients and range from non-life-threatening bleeding tosevere bleedings, such as intestinal and intracranial bleeding. Bleedingis the result of severe thrombocytopenia. Platelets of WAS and XLT havereduced size, which is the most common finding (100% incidence).Thrombocytopenia occurs irrespectively of the severity of the mutationand is possibly caused by instability of mutated WASp in platelets.Despite intensive research, the mechanisms underlying WASp-relatedthrombocytopenia and hemorrhages are not completely understood.Peripheral destruction of platelets in the spleen is thought to play animportant role in inducing thrombocytopenia because a substantialcorrection of the platelet count and size after splenectomy has beenreported. Overall, a full comprehension of the mechanisms causingthrombocytopenia still needs to be achieved.

In yet some more specific embodiments, the method of the invention maybe applicable for treating inflammatory chronic skin conditions,specifically, eczema. The typical skin lesions in WAS and XLT patientsresemble acute or chronic eczema in appearance and distribution. Eczemadevelops in 80% of the patients and is heterogeneous in severity andpersistence. Indeed, in its most severe form, eczema is resistant totherapy, persists into adulthood, and facilitates opportunistic skininfections (Molluscum contagiosum, herpes simplex, or bacteria). Theincidence and severity of eczema are significantly lower in patientswith residual WASp expression. The causes of eczema in WAS patients arecurrently unknown.

Still further embodiments, related to the method of the invention thatmay be suitable for treating autoimmune conditions. WAS-associatedautoimmune complications are frequently observed. The incidence ofautoimmunity in classic WAS is high in the US and European populations(40%-72%), whereas a lower incidence was reported in Japan (22%). Themost common manifestations are autoimmune hemolytic anemia, cutaneousvasculitis, arthritis, and nephropathy. Less common autoimmunemanifestations include inflammatory bowel disease, idiopathicthrombocytopenic purpura, and neutropenia. Patients frequently havemultiple autoimmune manifestations at the same time. Development ofautoimmunity can have a prognostic value. Indeed, it has been reportedthat WAS patients who develop autoimmune hemolytic anemia or autoimmunethrombocytopenia early (180 days) after splenectomy have a poorprognosis. Moreover, autoimmunity is associated with a higher risk of alater development of tumors and an increased risk of mortality. Untilnow, the mechanisms of WAS-associated autoimmunity have not beenclarified. It has been proposed that autoimmunity could be the result ofa bystander tissue damage originating from the chronic inflammatorystate that is established after incomplete pathogen clearance.

Still further embodiments relate to the applicability of the methods ofthe invention in the treatment of tumors in WAS patients. Recent surveysreported an increased tumor incidence in WAS patients. Tumors can ariseduring childhood (especially myelodysplasia) but are more frequent inadolescents and young adults. WAS-associated tumors are mainlylymphoreticular malignancies, with leukemia, myelodysplasia, andlymphoma resulting in up to 90% of the cases. WAS-associatedmalignancies have a poor prognosis because less than 5% of patientssurvive 2 years after diagnosis, and result in up to 25% of death cases.Immune deficiency can contribute to the genesis of tumors.

One of the known abnormalities associated with WAS are dysfunction ofdendritic cells (DC), which are bone marrow-derived antigen presentingcells that are required for the initiation of immune responses. Aprominent role for DC has been proposed in many clinical situationsincluding autoimmunity, transplantation, malignancy and vaccination. Itis probable that other molecularly undefined immunodeficiencies havedefects in this cell population, and that disruption of DC polarizationand motility, involving WASp could contribute to more commoninflammatory conditions such as atopic eczema and autoimmunity.Intrinsic dysfunction of the DC population may also have an importantrole in the pathogenesis of other primary immunodeficiency syndromes,while changes in DC cytoskeletal signaling pathways may contribute tothe initiation of acquired immunological and inflammatory disorders.

It was also found that WASP has a role in regulating IL-2 production,which is independent of its role in immune synapse formation. Therefore,disorders associated with impaired IL2 production may benefit from WASpupregulation. Thus, in some embodiments, the invention may furtherprovide methods for treating disorders associated with impaired IL-2production.

As noted above, acquired disorders associated with WASp dysfunction mayinclude autoimmune disorders, inflammatory disorders and atopic eczema.Therefore, in further embodiments the pharmaceutical compositions andmethods of the invention may be suitable for treatment of acquireddisorders related to WASp dysfunction.

Still further, in certain embodiments, the methods and compositions ofthe invention may be applicable for treating disorders associated withimmunodeficiency. ‘Immunodeficiency’, primary or secondary, meaninginherited or acquired, respectively. The term ‘immunodeficiency’ isintended to convey a state of an organism, wherein the immune system'sability for immuno-surveillance of infectious disease or cancer iscompromised or entirely absent.

According to the International Union of Immunological Societies, morethan 150 primary immunodeficiency diseases (PIDs) have beencharacterized, and the number of acquired (or secondary)immuno-deficiencies exceeds the number of PIDs. PIDs are those caused byinherited genetic mutations. Secondary immuno-deficiencies are caused byvarious conditions, aging or agents such as viruses or immunesuppressing drugs.

Thus, in certain embodiments, the methods of the invention may beapplicable for subjects suffering from secondary immunodeficiency. Inyet some further embodiments, such immunodeficiency may be caused by atleast one of chemotherapy, radiotherapy, biological therapy, bone marrowtransplantation, gene therapy, adoptive cell transfer or anycombinations thereof.

As indicated above, the in some further embodiments, the SMCs of theinvention may be applicable in boosting the immune response of a subjectsuffering from a secondary immunosuppression caused by chemotherapy,specifically, treatment with a chemotherapeutic agent.

“chemotherapeutic agent” or “chemotherapeutic drug” (also termedchemotherapy) as used herein refers to a drug treatment intended foreliminating or destructing (killing) cancer cells or cells of any otherproliferative disorder. The mechanism underlying the activity of somechemotherapeutic drugs is based on destructing rapidly dividing cells,as many cancer cells grow and multiply more rapidly than normal cells.As a result of their mode of activity, chemotherapeutic agents also harmcells that rapidly divide under normal circumstances, for example bonemarrow cells, digestive tract cells, and hair follicles. Insulting ordamaging normal cells result in the common side-effects of chemotherapy:myelosuppression (decreased production of blood cells, hence alsoimmuno-suppression), mucositis (inflammation of the lining of thedigestive tract), and alopecia (hair loss).

Various different types of chemotherapeutic drugs are available. Achemotherapeutic drug may be used alone or in combination with anotherchemotherapeutic drug or with other forms of cancer therapy, such as abiological drug, radiation therapy or surgery.

Certain chemotherapy agents have also been used in the treatment ofconditions other than cancer, including ankylosing spondylitis, multiplesclerosis, hemangiomas, Crohn's disease, psoriasis, psoriatic arthritis,rheumatoid arthritis, lupus and scleroderma.

Chemotherapeutic drugs affect cell division or DNA synthesis andfunction and can be generally classified into groups, based on theirstructure or biological function. The present invention generallypertains to chemotherapeutic agents that are classified as alkylatingagents, anti-metabolites, anthracyclines, plant alkaloids, topoisomeraseinhibitors, and other anti-tumor agents such as DNA-alkylating agents,anti-tumor antibiotic agents, tubulin stabilizing agents, tubulindestabilizing agents, hormone antagonist agents, protein kinaseinhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors,caspase inhibitors, metalloproteinase inhibitors, antisense nucleicacids, triple-helix DNAs, nucleic acids aptamers, andmolecularly-modified viral, bacterial or exotoxic agents.

However, several chemotherapeutic drugs may be classified as relating tomore than a single group. It is noteworthy that some agents, includingmonoclonal antibodies and tyrosine kinase inhibitors, which aresometimes referred to as “chemotherapy”, do not directly interfere withDNA synthesis or cell division but rather function by targeting specificcomponents that differ between cancer cells and normal cells and aregenerally referred to as “targeted therapies”, “biological therapy” or“immunotherapeutic agent” as detailed below.

More specifically, as their name implies, alkylating agents function byalkylating many nucleophilic functional groups under conditions presentin cells. Examples of chemotherapeutic agents that are considered asalkylating agents are cisplatin and carboplatin, as well as oxaliplatin.Alkylating agents impair cell function by forming covalent bonds withamino, carboxyl, sulfhydryl, and phosphate groups in variousbiologically-significant molecules. Examples of agents which function bychemically modifying DNA are mechlorethamine, cyclophosphamide,chlorambucil and ifosfamide. An additional agent acting as a cell cyclenon-specific alkylating antineoplastic agent is the alkyl sulfonateagent busulfan (also known as Busulfex). alkylating chemotherapeuticagent cyclophosphamide

In some particular embodiments, the immune-suppressive condition may becaused by treatment with oxaliplatin. More specifically, Oxaliplatin isa platinum-based chemotherapy drug in the same family as cisplatin andcarboplatin. It is typically administered in combination withfluorouracil and leucovorin in a combination known as Folfox for thetreatment of colorectal cancer. Compared to cisplatin the two aminegroups are replaced by cyclohexyldiamine for improved antitumouractivity. The chlorine ligands are replaced by the oxalato bidentatederived from oxalic acid in order to improve water solubility.Oxaliplatin is marketed by Sanofi-Aventis under the trademark Eloxatin®.

Still Further, anti-metabolites (also termed purine and pyrimidineanalogues) mimic the structure of purines or pyrimidines which are thebuilding blocks of DNA and may thus be incorporated into DNA. Theincorporation of anti-metabolites into DNA interferes with DNAsyntheses, leading to abnormal cell development and division.Anti-metabolites also affect RNA synthesis. Examples of anti-metabolitesinclude 5-fluorouracil (5-FU), azathioprine and mercaptopurine,fludarabine, cladribine (2-chlorodeoxyadenosine, 2-CdA), pentostatin(2′-deoxycoformycin, 2′-DCF), nelarabine, Floxuridine (FUDR),gemcitabine (Gemzar, a synthetic pyrimidine nucleoside) and Cytosinearabinoside (Cytarabine).

In yet some further embodiments, the SMCs of the invention may beapplicable for boosting an immune-response in a subject treated with achemotherapeutic agent that may be at least one Plant alkaloid andterpenoid. Plant alkaloids and terpenoids are agents derived from plantsthat block cell division by preventing microtubule function, therebyinhibiting the process of cell division (also known as “mitoticinhibitors” or “anti-mitotic agents”). Examples of alkaloids include thevinca alkaloids (e.g. vincristine, vinblastine, vinorelbine andvindesine) and terpenoids include, for example, taxanes (e.g. paclitaxeland docetaxel). Taxanes act by enhancing the stability of microtubules,preventing the separation of chromosomes during anaphase.

In further embodiments, the SMCs of the invention may be applicable forboosting an immune-response in a subject treated with chemotherapeuticagent that may be at least one Topoisomerase inhibitor. Topoisomerasesare essential enzymes that maintain DNA topology (i.e. the overall threedimensional structure of DNA). Inhibition of type I or type IItopoisomerases interferes with both transcription and replication of DNAby inhibiting proper DNA supercoiling. Type I topoisomerase inhibitorsinclude camptothecins [e.g. irinotecan and topotecan (CPT11)] andexamples of type II inhibitors include amsacrine, etoposide, etoposidephosphate, and teniposide.

Still further, Anthracyclines (or anthracycline antibiotics) are a classof drugs used in cancer chemotherapy that are derived from thestreptomyces bacterium. These compounds are used to treat many cancers,including leukemias, lymphomas, breast, uterine, ovarian, and lungcancers. These agents include, inter alia, the drugs daunorubicin (alsoknown as Daunomycin), and doxorubicin and many other related agents(e.g., Valrubicin and Idarubicin). For example, the anthracycline agentIdarubicin acts by interfering with the enzyme topoisomerase II.

In further embodiments, the SMCs of the invention may be applicable forboosting an immune-response in a subject treated with Doxorubicin. Thechemotherapeutic agent Doxorubicin (also known by the trade nameAdriamycin and by the name hydroxydaunorubicin) is an anthracyclineantibiotic that is closely related to the natural product daunomycin,and like all anthracyclines, it works by intercalating DNA. The mostserious adverse side effect of using this agent is the life-threateningheart damage. It is commonly used in the treatment of a wide range ofcancers, including hematological malignancies, many types of carcinoma,and soft tissue sarcomas.

In certain embodiments, the SMCs of the invention may be applicable forboosting an immune-response in a subject treated with chemotherapeuticagent that may be at least one Cytotoxic antibiotics. The anthracyclinesagents described above are also classified as “cytotoxic antibiotics”.Cytotoxic antibiotics also include the agent actinomycin D (also knowngenerically as Actinomycin or Dactinomycin), which is the mostsignificant member of the actinomycines class of polypeptide antibiotics(that were also isolated from streptomyces). Actinomycin D is shown tohave the ability to inhibit transcription by binding DNA at thetranscription initiation complex and preventing elongation of RNA chainby RNA polymerase. Other cytotoxic antibiotics include bleomycin,epirubicin and mitomycin.

Still further, in some embodiments the SMCs of the invention may beapplicable for subjects suffering from immune-deficiency caused byimmune-therapy or a biological therapy. More specifically, cancervaccines, antibody treatments, and other “immunotherapies” arepotentially more specific and effective and less toxic than the currentapproaches of cancer treatment and are generally termed “immunotherapy”,and therefore, an agent used for immunotherapy, is defined herein as animmuno-therapeutic agent. The term immunotherapy as herein defined (alsotermed biologic therapy or biotherapy) is a treatment that uses certaincomponents of the immune system to fight diseases (e.g. cancer), by,inter alia, stimulating the immune system to become more efficient inattacking cancer cells (e.g., by administering vaccines) or byadministering components of the immune system (e.g., by administeringcytokines, antibodies, etc.).

In the last few decades immunotherapy has become an important part oftreating several types of cancer with the main types of immunotherapyused being monoclonal antibodies (either naked or conjugated), cancervaccines (i.e. that induce the immune system to mount an attack againstcancer cells in the body) and non-specific immunotherapies.

Antibody-mediated therapy as referred to herein refers to the use ofantibodies that are specific to a cancer cell or to any protein derivedthere-from for the treatment of cancer. As a non-limiting example, suchantibodies may be monoclonal or polyclonal which may be naked orconjugated to another molecule. Antibodies used for the treatment ofcancer may be conjugated to a cytotoxic moiety or radioactive isotope,to selectively eliminate cancer cells.

It should be noted that the term “biological treatment” or “biologicalagent”, as used herein refers to any biological material that affectsdifferent cellular pathways. Such agent may include antibodies, forexample, antibodies directed to cell surface receptors participating insignaling, that may either activate or inhibit the target receptor. Suchbiological agent may also include any soluble receptor, cytokine,peptides or ligands. Non limiting examples for monoclonal antibodiesthat are used for the treatment of cancer include bevacizumab (alsoknown as Avastin), rituximab (anti CD20 antibody), cetuximab (also knownas Erbitux), anti-CTLA4 antibody and panitumumab (also known asVectibix) and anti Gr1 antibodies.

More specifically, cancer vaccines as referred to herein are vaccinesthat induce the immune system to mount an attack against cancer cells inthe body. A cancer treatment vaccine uses cancer cells, parts of cells,or pure antigens to increase the immune response against cancer cellsthat are already in the body. These cancer vaccines are often combinedwith other substances or adjuvants that enhance the immune response.

Non-specific immunotherapies as referred to herein do not target acertain cell or antigen, but rather stimulate the immune system in ageneral way, which may still result in an enhanced activity of theimmune system against cancer cells. A non-limiting example ofnon-specific immunotherapies includes cytokines (e.g. interleukins,interferons). It should be thus appreciated that in some embodiments,the SMCs of the invention may be used as a combined supportive treatmentfor patients suffering from immune suppression. This supportivetreatment may be combined with other supportive therapies as discussedherein.

Thus, in yet other embodiments, SMCs of the invention may be applicablefor subjects undergoing at least one of adoptive cell transfer, a cancervaccine, antibody-based therapy, a hormone, a cytokine or anycombination thereof.

As indicated above, in some embodiments, the SMCs of the invention maybe used for hematopoietic cell reconstitution in subjects undergoingradiotherapy. Radiation therapy or radiotherapy, often abbreviated RT,RTx, or XRT, is therapy using ionizing radiation, generally as part ofcancer treatment to control or kill malignant cells and normallydelivered by a linear accelerator. Radiation therapy may be curative ina number of types of cancer if they are localized to one area of thebody. It may also be used as part of adjuvant therapy, to prevent tumorrecurrence after surgery to remove a primary malignant tumor (forexample, early stages of breast cancer). According to some specificembodiment, the radiation is ionizing radiation, which may be any one ofX-rays, gamma rays and charged particles. In other embodiments, theradiation may be employed in the course of total body irradiation,brachytherapy, radioisotope therapy, external beam radiotherapy,stereotactic radio surgery (SRS), stereotactic body radiation therapy,particle or proton therapy, or body imaging using the ionizingradiation.

As indicated above, in some embodiments, the SMCs of the invention maybe used for hematopoietic cell reconstitution in subjects undergoinggene therapy. Gene therapy is the therapeutic delivery of nucleic acidpolymers into a patient's cells as a drug to treat disease. The mostcommon form uses DNA, optionally packed in a vector, that encodes afunctional, therapeutic gene to replace a mutated gene.

In yet some further embodiments, the SMCs of the invention may be usedfor enhancing reconstitution of leukocyte and megakaryocyte populationsin a subject that undergoes adoptive transfer. The term “adoptivetransfer” as herein defined applies to all the therapies that consist ofthe transfer of components of the immune system that are already capableof mounting a specific immune response. Examples of adoptive transferinclude both the transfer of antibodies and also, in adoptive celltransfer, specific types of cells that are capable of mediatingantigen-specific tumor regression such as LAK and T cells. Cell-basedtherapies with various lymphocytes and antigen-presenting cells arepromising approaches for cancer immunotherapy. The transfusion of Tlymphocytes, also called adoptive cell therapy (ACT), is an effectivetreatment for viral infections and has induced regression of cancer inearly stage clinical trials.

As noted above, conditions alleviated or modulated by the administrationof the SMCs are typically those characterized by a reduced hematopoieticor immune function and more specifically, a reduced neutrophil count.Such conditions may be induced as a course of therapy for otherpurposes, such as chemotherapy or radiation therapy. Such conditions mayresult from infectious disease, such as bacterial, viral, fungal, orother infectious disease. For example, sepsis results from bacterialinfection. Or, such condition may be hereditary or environmentallycaused, such as severe chronic neutropenia. Age may also play a factor,as in the geriatric setting; patients may have a reduced neutrophilmobilization, reduced hematopoietic function, reduced immune function,reduced neutrophil count, reduced platelets count, sepsis, severechronic neutropenia, infectious diseases, leukopenia, thrombocytopenia,anemia, enhancing engraftment of bone marrow during transplantation,enhancing bone marrow recovery in treatment of radiation, chemical orchemotherapeutic induced bone marrow aplasia or myelosuppression, andacquired immune deficiency syndrome.

Leukopenia is a decrease in the number of white blood cells (leukocytes)found in the blood, which places individuals at increased risk ofinfection. Neutropenia, a subtype of leukopenia, refers to a decrease inthe number of circulating neutrophil granulocytes, the most abundantwhite blood cells. Low white cell count may be due to acute viralinfections. It has been associated with chemotherapy, radiation therapy,myelofibrosis, aplastic anemia (failure of white cell, red cell andplatelet production), stem cell transplant, bone marrow transplant,AIDS, and steroid use.

Other causes of low white blood cell count include systemic lupuserythematosus, Hodgkin's lymphoma and some types of cancer, typhoid,malaria, tuberculosis, dengue, rickettsial infections, enlargement ofthe spleen, folate deficiencies, psittacosis, sepsis, Sjögren's syndromeand Lyme disease. It has also been shown to be caused by deficiency incertain minerals, such as copper and zinc. The SMCs of the invention maybe applicable for any of the conditions disclosed above.

In some further embodiments, the methods of the invention may beapplicable for immune-related disorder or condition that may be apathologic condition caused by at least one pathogen.

It should be appreciated that an infectious disease as used herein alsoencompasses any infectious disease caused by a pathogenic agent,specifically, a pathogen. Pathogenic agents include prokaryoticmicroorganisms, lower eukaryotic microorganisms, complex eukaryoticorganisms, viruses, fungi, prions, parasites, yeasts, toxins and venoms.

A prokaryotic microorganism includes bacteria such as Gram positive,Gram negative and Gram variable bacteria and intracellular bacteria.Examples of bacteria contemplated herein include the species of thegenera Treponema sp., Borrelia sp., Neisseria sp., Legionella sp.,Bordetella sp., Escherichia sp., Pseudomonas sp. (e.g., P. aeruginosa),Salmonella sp., Shigella sp., Klebsiella sp., Yersinia sp., Vibrio sp.,Hemophilus sp., Rickettsia sp., Chlamydia sp., Mycoplasma sp.,Staphylococcus sp., Streptococcus sp., (e.g., Streptococcus pneumonia),Bacillus sp., Clostridium sp., Corynebacterium sp., Proprionibacteriumsp., Mycobacterium sp., (e.g., Mycobacteria tuberculosis), Ureaplasmasp. and Listeria sp.

A lower eukaryotic organism includes a yeast or fungus such as but notlimited to Pneumocystis carinii, Candida albicans, Aspergillus,Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcusneoformans, Trichophyton and Microsporum.

A complex eukaryotic organism includes worms, insects, arachnids,nematodes, aemobe, Entamoeba histolytica, Giardia lamblia, Trichomonasvaginalis, Trypanosoma brucei gambiense, Trypanosoma cruzi, Balantidiumcoli, Toxoplasma gondii, Cryptosporidium or Leishmania.

In yet some further embodiments, the SMCs of the invention may beapplicable in boosting the immune response against a pathogen that maybe in further specific embodiment, a viral pathogen or a virus. The term“viruses” is used in its broadest sense to include viruses of thefamilies adenoviruses, papovaviruses, herpesviruses: simplex,varicella-zoster, Epstein-Barr (EBV), Cytomegalo virus (CMV), poxviruses: smallpox, vaccinia, hepatitis B (HBV), rhinoviruses, hepatitisA (HBA), poliovirus, rubella virus, hepatitis C (HBC), arboviruses,rabies virus, influenza viruses A and B, measles virus, mumps virus,human deficiency virus (HIV), HTLV I and II and Zika virus.

Of particular relevance for acquired immunodeficiency caused by a viralpathogen, is any immunodeficiency caused by HIV. More specifically, insome further embodiments, the SMCs of the invention may be applicablefor AIDS. Acquired immunodeficiency syndrome (AIDS) is defined in termsof either a CD4+ T cell count below 200 cells per L or the occurrence ofspecific diseases in association with an HIV infection. In the absenceof specific treatment, around half of people infected with HIV developAIDS within ten years. The most common initial conditions that alert tothe presence of AIDS are pneumocystis pneumonia (40%), cachexia in theform of HIV wasting syndrome (20%), and esophageal candidiasis. Othercommon signs include recurring respiratory tract infections.

In yet some further embodiments, the SMCs of the invention may beapplicable in boosting the immune response against a pathogen that maybe a fungal pathogen. The term “fungi” includes for example, fungi thatcause diseases such as ringworm, histoplasmosis, blastomycosis,aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis,paracoccidio-idoinycosis, and candidiasis.

Still further, the term parasite includes, but not limited to,infections caused by somatic tapeworms, blood flukes, tissue roundworms,ameba, and Plasmodium, Trypanosoma, Leishmania, and Toxoplasma species.

In certain additional embodiments, the methods of the invention may beapplicable for immune-related disorder or condition that may bethrombocytopenia. More particularly, thrombocytopenia cause by bleeding,chemo- or radiotherapy, or autoimmunity (ITP).

The proposed therapeutic approaches can also benefit several orphandiseases such as Idiopathic thrombocytopenic purpura (ITP). ITP is atype of thrombocytopenic purpura defined as isolated low platelet count(thrombocytopenia) with normal bone marrow and the absence of othercauses of thrombocytopenia. It causes a characteristic purpuric rash andan increased tendency to bleed. ITP is an autoimmune disease withantibodies detectable against several platelet surface antigens. ITP isdiagnosed by a low platelet count in a complete blood count (a commonblood test).

As shown by the examples, the SMCs of the invention induce plateletsactivation and as such may modulate and enhance coagulation processes.Induction of coagulation may be applicable in prevention andameliorating conditions that may involve bleeding. Thus, in someembodiments, the SMCs of the invention may be applicable for treatingacquired hemostatic disorders. The acquired hemostatic disorder may beat least one of surgery-induced bleeding, trauma-induced bleeding, acutegastrointestinal bleeding, bleeding associated with burns, hemorrhagicstroke, lung injury associated with emphysema and chronic obstructivepulmonary disease (COPD), bleeding associated with childbirth,disseminated intravascular coagulation (DIC), and bleeding resultingfrom fibrinolytic or thrombolytic therapy.

In some specific embodiments, the SMCs of the invention may beapplicable for treating, preventing, reducing, attenuating, andinhibiting bleeding associated with surgical procedures, specifically,minor or major surgical procedures.

In a further specific embodiment the method of the invention may besuitable for treating trauma-induced bleeding (traumatic bleeding).Traumatic bleeding can be caused by any type of injury, for example anyinjury caused by, work and car accidents, combats or falls. There aredifferent types of traumatic wounds which may cause bleeding. Ingeneral, trauma causes damage to a blood vessels that in turn causesblood to flow externally outside the body or internally into body organssuch as brain, lung, liver, kidney, spleen or into body cavities, suchas thorax and abdomen.

In some specific embodiments the SMCs of the invention may be suitablefor treatment of acute or chronic gastrointestinal bleeding, that mayinclude upper gastrointestinal bleeding and lower gastrointestinalbleeding.

In yet further embodiments the SMCs of the invention may be applicablefor the treatment of hemorrhagic stroke or any other brain injury ortrauma.

In certain specific embodiments, the SMCs the invention may be suitablefor treating, preventing, reducing, attenuating, and inhibiting bleedingassociated with surgical intervention, specifically, a minor or a majorsurgery. More specifically, it should be understood that in cases thesurgical procedures are elective, expected or not urgent (e.g., cesareansurgery, or any other major surgery that allow sufficient time forpre-operative preparations), the SMCs of the invention may be used forpre-operative treatment to facilitate prevention or reduction ofexcessive bleeding during the surgical intervention.

Major surgery is defined as any surgical procedure that involvesanesthesia or respiratory assistance. In more specific embodiments,major surgery may be an open heart surgery. In yet some otherembodiments a major surgery may be liver transplantation surgery.

It should be further recognized that the treatment with the SMCs of theinvention may be particularly applicable for treating the bleedingmanifestations induced by thrombolytic/fibrinolytic therapy. As usedherein, the term “anticoagulant agent” is intended to mean any agentwhich interferes with the clotting of blood. Some anticoagulants, suchas the coumarin derivatives bishydroxycoumarin (Dicumarol) and warfarin(Coumadin) inhibit synthesis of prothrombin, a clot-forming substance,and other clotting factors. Anticoagulants can include but are notlimited to compounds acting as beta2 Adrenoreceptor Antagonists,Neuropeptide V2 Antagonists, prostacyclin analogs, thromboxane synthaseinhibitors, calcium agonists, coumarin derivatives, elastase inhibitors,Non-steroidal anti-inflammatories thrombin inhibitors, lipoxygenaseinhibitors, Factor Vila inhibitors, Factor Xa inhibitors,phosphodiesterase III inhibitors, Heparins, and fibrinogen glucoproteinIIb/IIIa Antagonists.

SMC-based treatment may also be used to enhance anti-tumor immunity.CTLs, as well as NK cells serve as the primary mediators of immuneresponse against cancer cells. WASp was shown to be essential forNK-cancer cell conjugate formation and cytotoxic capacity. NK cells fromWAS patients exhibit defective cytotoxicity. Thus, WASp enhancing SMCsmay increase the potency of this response by improving the ability ofthe cells to migrate into the tumor, as well as improving activation andcytotoxic granule release.

The term “anti-tumor immunity” refers to innate and adaptive immuneresponses which may lead to tumor control.

The immune system can be activated by tumor antigens and, once primed,can elicit an antitumor response. Activated tumor specific cytotoxic Tlymphocytes (CTLs) can seek out and destroy metastatic tumor cells andreduce tumor lesions. Natural Killer (NK) cells are a front-line defenseagainst drug-resistant tumors and can provide tumoricidal activity toenhance tumor immune surveillance. Cytokines like IFN-γ or TNF play acrucial role in creating an immunogenic microenvironment and thereforeare key players in the fight against metastatic cancer. Critical aspectsin the tumor-immune system interface include the processing andpresentation of released antigens by antigen-presenting cells (APCs),interaction with T lymphocytes, subsequent immune/T-cell activation,trafficking of antigen-specific effector cells, and, ultimately, theengagement of the target tumor cell by the activated effector T cell.

Nevertheless, although often successful in preventing tumor outgrowth,this “cancer-immunity cycle” can be disrupted by artifices involved inimmune escape and development of tolerance, culminating with the evasionand proliferation of malignant cells.

Furthermore, the tumor microenvironment induces suppression and reducedactivity of NK and T cells, through the secretion of inhibitory factorssuppressing the anti-tumor response, a phenomena known as exhaustion.Increasing WASp, a mediator of activating immune signaling maycounteract this increased inhibitory signaling and allow a robust T celland NK cell mediated cytotoxic response.

In still some further embodiments, the methods of the invention may beapplicable for immune-related disorder or condition that may be acancer. In some embodiments, such cancer may be either of anon-hematopoietic origin or alternatively, of a hematological origin. Itshould be appreciated that is some embodiments, the methods of theinvention may be used as a supportive treatment for boosting theimmune-system, specifically, cytotoxic lymphocytes activity.

As used herein to describe the present invention, “cancer”,“proliferative disorder”, “tumor” and “malignancy” all relateequivalently to a hyperplasia of a tissue or organ. If the tissue is apart of the lymphatic or immune systems, malignant cells may includenon-solid tumors of circulating cells. Malignancies of other tissues ororgans may produce solid tumors. In general, the SMC modulators andmethods of the present invention may be applicable for the treatment ofa patient suffering from any one of non-solid and solid tumors.

Malignancy, as contemplated in the present invention may be any one ofcarcinomas, melanomas, sarcomas and also in some embodiments, mayinclude lymphomas, leukemia and myeloma.

More specifically, carcinoma as used herein refers to an invasivemalignant tumor consisting of transformed epithelial cells.Alternatively, it refers to a malignant tumor composed of transformedcells of unknown histogenesis, but which possess specific molecular orhistological characteristics that are associated with epithelial cells,such as the production of cytokeratins or intercellular bridges.

Melanoma as used herein, is a malignant tumor of melanocytes.Melanocytes are cells that produce the dark pigment, melanin, which isresponsible for the color of skin. They predominantly occur in skin, butare also found in other parts of the body, including the bowel and theeye. Melanoma can occur in any part of the body that containsmelanocytes.

Sarcoma is a cancer that arises from transformed connective tissuecells. These cells originate from embryonic mesoderm, or middle layer,which forms the bone, cartilage, and fat tissues. This is in contrast tocarcinomas, which originate in the epithelium. The epithelium lines thesurface of structures throughout the body, and is the origin of cancersin the breast, colon, and pancreas.

Further malignancies that may find utility in the present invention cancomprise but are not limited to various solid tumors including GI tract,colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma. Theinvention may be applicable as well for the treatment or inhibition ofsolid tumors such as tumors in lip and oral cavity, pharynx, larynx,paranasal sinuses, major salivary glands, thyroid gland, esophagus,stomach, small intestine, colon, colorectum, anal canal, liver,gallbladder, extraliepatic bile ducts, ampulla of vater, exocrinepancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma,carcinoma and malignant melanoma of the skin, breast, vulva, vagina,cervix uteri, corpus uteri, ovary, fallopian tube, gestationaltrophoblastic tumors, penis, prostate, testis, kidney, renal pelvis,ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma ofthe conjunctiva, malignant melanoma of the conjunctiva, malignantmelanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland,sarcoma of the orbit, brain, spinal cord, vascular system,hemangiosarcoma and Kaposi's sarcoma.

As noted above, in some specific embodiments, the SMC modulators of theinvention may be applicable in upregulating the immune-response againstcancers of non-hematopoietic origin. Specifically, any of the canerdisorders disclosed above. More specifically, cancer that is of anon-hematologic origin or non-hematological cancer or malignancy may beany cancer originated from any cell that is not of a lymphoid or myeloidorigin. In some embodiments however, the SMCs of the invention may beapplicable for hematological cancers, specifically, leukemia, lymphomaand myeloma.

More specifically, leukemia refers to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number of abnormal cells in the blood-leukemic or aleukemic(subleukemic).

Myeloma as mentioned herein is a cancer of plasma cells, a type of whiteblood cell normally responsible for the production of antibodies.Collections of abnormal cells accumulate in bones, where they cause bonelesions, and in the bone marrow where they interfere with the productionof normal blood cells. Most cases of myeloma also feature the productionof a paraprotein, an abnormal antibody that can cause kidney problemsand interferes with the production of normal antibodies leading toimmunodeficiency. Hypercalcemia (high calcium levels) is oftenencountered.

Lymphoma is a cancer in the lymphatic cells of the immune system.Typically, lymphomas present as a solid tumor of lymphoid cells. Thesemalignant cells often originate in lymph nodes, presenting as anenlargement of the node (a tumor). It can also affect other organs inwhich case it is referred to as extranodal lymphoma. The term lymphomaas used herein encompasses T cell as well as B cell lymphoma. Nonlimiting examples for lymphoma include Hodgkin's disease, non-Hodgkin'slymphomas and Burkitt's lymphoma.

Hematological malignancies (including lymphoma, leukemia andmyeloproliferative disorders, as described above), may further includehypoplastic and aplastic anemia (both virally induced and idiopathic),myelodysplastic syndromes, all types of paraneoplastic syndromes (bothimmune mediated and idiopathic).

In yet some further embodiments, where the SMCs of the invention areused for boosting the immune-system of a subject that suffers of asecondary-immunosuppression caused by chemo- or radiotherapy. In morespecific embodiments, such radio- or chemotherapy may be applied on asubject suffering from a malignant disorder. In yet some furtherembodiments the subject may be affected by a non-hematopoieticmalignancy. In yet some further embodiments, the subject may be affectedby a hematopoietic malignancy. It should be noted that any of themalignancies discussed herein above are relevant for any aspect of theinvention and for any use of the SMCs, specifically, any of the usesdisclosed by the invention.

In yet some further embodiments, embodiments, the methods of theinvention may be applicable for a subject that suffers from a cancer andundergoes at least one of chemotherapy, radiotherapy, biologicaltherapy, or any combinations thereof. In some specific embodiments, suchsubject may be suffering from a non-hematologic cancer and undergoes achemotherapy, radiotherapy, a biological therapy or any combinationsthereof. Alternatively, the subject may be suffering from hematologiccancer and undergoes a chemotherapy, radiotherapy, a biological therapyor any combinations thereof. In yet some further specific embodiments,such method may further comprise the step of administering to thetreated subject before, simultaneously with, after or any combinationthereof, the administration of the SMC modulator/s of the invention, atleast one agent that induces differentiation of hematopoietic progenitorcells. In some specific and non-limiting embodiments, such treatment maybe any supportive treatment. In yet some further particular embodiments,such treatment may include G-CSF and/or any analogs thereof.

As indicated above, the methods of the invention may be used in someembodiments, for treating any autoimmune disorder. It should be notedthat an “autoimmune disorder” is a condition associated with dysfunctionof the immune system of a subject, either through activation orsuppression of the immune system or that can be treated, prevented ordiagnosed by targeting a certain component of the immune response in asubject, such as the adaptive or innate immune response. Such disordermay be any one of an inflammatory disease or an autoimmune disease.

In other embodiments, the pharmaceutical compositions and methods of theinvention can be suitable for treatment of inflammatory disorders. Basedon the insufficient availability of specific anti-inflammatorytreatments and the awareness of the roles of WASp in the pathogenesis ofseveral groups of disorders, the development of new anti-inflammatorytreatments is anticipated. Thus, there is a current need for theemployment of the present invention in therapy.

The general term “inflammatory disorder” relates to disorders where aninflammation is a main response to harmful stimuli, such as pathogens,damaged cells, or irritants. Inflammation is a protective response thatinvolves immune cells, blood vessels, and molecular mediators, as wellas the end result of long-term oxidative stress. More specifically,inflammatory disorders are a large group of disorders that underlie avast variety of human diseases. Also, the immune system can be involvedin inflammatory disorders, stemming from abnormal immune response of theorganism against substances of its own, or initiation the inflammatoryprocess for unknown reason, i.e. autoimmune and auto-inflammatorydisorders, respectively. Non-immune diseases with etiological origins ininflammatory processes include cancer, atherosclerosis, and ischemicheart disease.

The term “inflammatory disorders associated with WASp dysfunction” asused herein relates to at least one but not limited to the following:arthritis (ankylosing spondylitis, systemic lupus erythematosus,osteoarthritis, rheumatoid arthritis, psoriatic arthritis), asthma,atherosclerosis, inflammatory bowel disease (Crohn's disease, ulcerativecolitis) and dermatitis (including psoriasis).

In more specific embodiments, the SMC modulators of the invention or anypharmaceutical compositions thereof, as well as methods of the inventionmay be applicable for preventing, treating, ameliorating or inhibitinginflammatory bowel disease (IBD), specifically, ulcerative colitis andCrohn's disease.

Inflammatory bowel diseases (IBD) are common gastrointestinal disorders,that can be perceived as being the result of a disbalance betweenTh1-pro-inflammatory and Th2-anti-inflammatory subtypes of immuneresponses. IBD is a group of inflammatory conditions of the colon andsmall intestine. The major types of IBD are Crohn's disease andulcerative colitis (UC). Other forms of IBD account for far fewer cases.These are collagenous colitis, lymphocytic colitis, ischemic colitis,diversion colitis, and indeterminate colitis, in cases where it isimpossible to make a definitive diagnosis distinguishing Crohn's diseasefrom ulcerative colitis.

The main difference between Crohn's disease and UC is the location andnature of the inflammatory changes. Crohn's disease can affect any partof the gastrointestinal tract, from mouth to anus (skip lesions),although a majority of the cases start in the terminal ileum. Ulcerativecolitis, in contrast, is restricted to the colon and the rectum.Microscopically, ulcerative colitis is restricted to the mucosa(epithelial lining of the gut), while Crohn's disease affects the wholebowel wall. Finally, Crohn's disease and ulcerative colitis present withextra-intestinal manifestations (such as liver problems, arthritis, skinmanifestations and eye problems) in different proportions. Crohn'sdisease and ulcerative colitis share the same symptoms such as diarrhea,vomiting, weight loss, fever and abdominal pain.

According to other unlimited embodiments, the SMC modulators,compositions, and methods of the invention may be used for preventing,treating, ameliorating or inhibiting atopic eczema.

“Atopic eczema”, also known as atopic dermatitis, is the most commonform of eczema. It mainly affects children, but can also affect adults.Eczema is a condition that causes the skin to become itchy, red, dry andcracked. It is a long-term (chronic) condition in most people, althoughit can improve over time, especially in children. Atopic eczema canaffect any part of the body, but the most common areas to be affectedare: backs or fronts of the knees, outside or inside of the elbows,around the neck, hands, cheeks and scalp. The exact cause of atopiceczema is unknown, but it's clear it's not down to one single thing. Itoften occurs in people who get allergies—“atopic” means sensitivity toallergens. There is currently no cure for atopic eczema, but symptomatictreatment can help relieve the symptoms and many cases improve overtime. However, severe eczema often has a significant impact on dailylife and may be difficult to cope with physically and mentally.

In yet another aspect thereof, the invention provides the use of aneffective amount of at least one SMC modulator of WASp having thegeneral formula (I)

or a pharmaceutically acceptable salt, solvate, esters, hydrate,stereoisomer or physiologically functional derivative thereof, or anycombination thereof, or any vehicle, matrix, nano- or micro-particlecomprising the same, in the preparation of a composition or medicamentfor treating, preventing, inhibiting, reducing, eliminating, protectingor delaying the onset of an innate or acquired immune-related disorderor condition in a subject in need thereof, wherein

R₁ and R₂ are each independently from each other selected from H,straight or branched C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl,straight or branched C₁-C₁₂ alkoxy, a ring system containing five totwelve atoms, each optionally substituted by at least one of halide,hydroxyl, ester, ether, amide, amine, nitro, —C(═O)—O—(CH₂)_(n)—CH₃, R₅,or —NH—C(═O)—R₅, R₅ is an a ring system containing five to twelve atomsoptionally substituted by at least one halide or straight or branchedC₁-C₅ alkyl;

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to twelve membered saturated or unsaturated ring that mayoptionally include N, O, S, NH, C═N, C═O S═O, or SO₂ and may beoptionally be substituted with at least one of straight or branchedC₁-C₅ alkyl, hydroxyl, halide and cyano;

L1 and L2 are each independently from each other be absent or selectedfrom —(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—,—(CH₂)_(n)—O—, S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n),—(CH₂)_(n)—N—C(═O)—, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—, —NH—(CH₂)_(n)—,—C(═O)—NH—(CH₂)_(n)—; —S—S—(CH₂)_(n)—; —O—(CH₂)_(n)—; —NH—(CH₂)_(n)—;C(═O)—(CH₂)_(n)—; —S—(CH₂)_(n)—; —NH—S(═O)_(n)—(CH₂)_(n)—,—CH₂—S—C(O)—NH—CH₂—, —(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—(NH)_(n)—C(═O)—, —(CH₂)_(n)—N—C(═O)— L1and L2 may be are each independently from each other optionallysubstituted with C₁-C₅ alkyl, a ring system containing five to twelveatoms substituted with C₁-C₅ alkyl

each n, is an integer being independently from each other selected frombe 0 to 5;

R₃ and R₄ are each independently from each other be absent or selectedfrom a ring system containing five to 15 atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), (O)₂, —C(O)—CH₃, —C(O)—O—CH₃, halide, CF₃, nitro, amide, or R₅, R₅is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight or branched C₁-C₅ alkyl.

Still further, the invention in some other aspects thereof, provides theuse of an effective amount of at least one SMC modulator of WASp havingthe general formula (XI):

or a pharmaceutically acceptable salt, solvate, esters, hydrate,stereoisomer or physiologically functional derivative thereof, or anycombination thereof, or any vehicle, matrix, nano- or micro-particlecomprising the same, in the preparation of a composition for treating,preventing, inhibiting, reducing, eliminating, protecting or delayingthe onset of an innate or acquired immune-related disorder or conditionin a subject in need thereof. In more specific embodiments,

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other may be absent orselected to be absent or from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—,—(CH₂)—, —(CH₂)—O—, —NH—(CH₂)—, and each optionally substituted withC₁-C₅ alkyl, a ring system containing five to twelve atoms optionallysubstituted with C₁-C₅ alkyl;

R₃ and R₄ are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl.

In more specific embodiments, the invention provides the use of any ofthe SMC modulators as defined by the invention and described hereinbefore or of any combinations thereof.

As used herein, “disease”, “disorder”, “condition” and the like, as theyrelate to a subject's health, are used interchangeably and have meaningsascribed to each and all of such terms. It should be appreciated thatthe invention provides therapeutic methods applicable for any of thedisorders disclosed above, as well as to any condition or diseaseassociated therewith. It is understood that the interchangeably usedterms “associated”, “linked” and “related”, when referring topathologies herein, mean diseases, disorders, conditions, or anypathologies which at least one of: share causalities, co-exist at ahigher than coincidental frequency, or where at least one disease,disorder condition or pathology causes the second disease, disorder,condition or pathology. More specifically, as used herein, “disease”,“disorder”, “condition”, “pathology” and the like, as they relate to asubject's health, are used interchangeably and have meanings ascribed toeach and all of such terms.

The terms “effective amount” or “sufficient amount” mean an amountnecessary to achieve a selected result. The “effective treatment amount”is determined by the severity of the disease in conjunction with thepreventive or therapeutic objectives, the route of administration andthe patient's general condition (age, sex, weight and otherconsiderations known to the attending physician).

The terms “treat, treating, treatment” as used herein and in the claimsmean ameliorating one or more clinical indicia of disease activity byadministering a pharmaceutical composition of the invention in a patienthaving a pathologic disorder.

“The term “treatment” as used herein refers to the administering of atherapeutic amount of the composition of the present invention which iseffective to ameliorate undesired symptoms associated with a disease, toprevent the manifestation of such symptoms before they occur, to slowdown the progression of the disease, slow down the deterioration ofsymptoms, to enhance the onset of remission period, slow down theirreversible damage caused in the progressive chronic stage of thedisease, to delay the onset of said progressive stage, to lessen theseverity or cure the disease, to improve survival rate or more rapidrecovery, or to prevent the disease form occurring or a combination oftwo or more of the above.

The term “amelioration” as referred to herein, relates to a decrease inthe symptoms, and improvement in a subject's condition brought about bythe compositions and methods according to the invention, wherein saidimprovement may be manifested in the forms of inhibition of pathologicprocesses associated with the immune-related disorders described herein,a significant reduction in their magnitude, or an improvement in adiseased subject physiological state.

The term “inhibit” and all variations of this term is intended toencompass the restriction or prohibition of the progress andexacerbation of pathologic symptoms or a pathologic process progress,said pathologic process symptoms or process are associated with.

The term “eliminate” relates to the substantial eradication or removalof the pathologic symptoms and possibly pathologic etiology, optionally,according to the methods of the invention described below.

The terms “delay”, “delaying the onset”, “retard” and all variationsthereof are intended to encompass the slowing of the progress and/orexacerbation of a pathologic disorder or an infectious disease and theirsymptoms slowing their progress, further exacerbation or development, soas to appear later than in the absence of the treatment according to theinvention.

More specifically, treatment or prevention include the prevention orpostponement of development of the disease, prevention or postponementof development of symptoms and/or a reduction in the severity of suchsymptoms that will or are expected to develop. These further includeameliorating existing symptoms, preventing-additional symptoms andameliorating or preventing the underlying metabolic causes of symptoms.It should be appreciated that the terms “inhibition”, “moderation”,“reduction” or “attenuation” as referred to herein, relate to theretardation, restraining or reduction of a process by any one of about1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about95% to 99%, or about 99% to 99.9%.

With regards to the above, it is to be understood that, where provided,percentage values such as, for example, 10%, 50%, 120%, 500%, etc., areinterchangeable with “fold change” values, i.e., 0.1, 0.5, 1.2, 5, etc.,respectively.

The present invention as defined by the claims, the contents of whichare to be read as included within the disclosure of the specification,and will now be described by way of example with reference to theaccompanying Figures.

The present invention relates to the treatment of subjects, or patients,in need thereof. By “patient” or “subject in need” it is meant anyorganism who may be infected by the above-mentioned pathogens, and towhom the preventive and prophylactic kit/s, system/s and methods hereindescribed is desired, including humans, domestic and non-domesticmammals such as canine and feline subjects, bovine, simian, equine andmurine subjects, rodents, domestic birds, aquaculture, fish and exoticaquarium fish. It should be appreciated that the treated subject may bealso any reptile or zoo animal. More specifically, the kit/s andmethod/s of the invention are intended for preventing pathologiccondition in mammals. By “mammalian subject” is meant any mammal forwhich the proposed therapy is desired, including human, equine, canine,and feline subjects, most specifically humans. It should be noted thatspecifically in cases of non-human subjects, the method of the inventionmay be performed using administration via injection, drinking water,feed, spraying, oral lavage and directly into the digestive tract ofsubjects in need thereof. The present invention relates to the treatmentof subjects, or patients, in need thereof. It should be further notedthat particularly in case of human subject, administering of thecompositions of the invention to the patient includes bothself-administration and administration to the patient by another person.

In yet a further aspect, the invention provides a SMC modulator of WASpdegradation having the general formula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other may be absent orselected from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—, —(CH₂)—, —(CH₂)—O—,—NH—(CH₂)—, and each optionally substituted with C₁-C₅ alkyl, a ringsystem containing five to twelve atoms optionally substituted with C₁-C₅alkyl;

R₃ and R₄ are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl.

In some particular embodiments, specific and non-limiting examples ofthe SMC modulators of the invention, may be pharmaceutically acceptablesalts or hydrates of the compounds of Formula XI, or in some furtherspecific embodiments, pharmaceutically acceptable salts or hydrates ofthe compounds of Formula XII. More specifically, such compounds mayinclude:

1-(2,6-Dimethyl-piperidin-1-yl)-2-[5-(quinolin-8-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-ethanone(designated herein as SMC 34); or

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-3-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(designated herein as SMC 34.7).

In yet some further particular embodiments, specific and non-limitingexamples of the SMC modulators of the invention, or pharmaceuticallyacceptable salts or hydrates of the compounds of Formula XI, or in somefurther specific embodiments, pharmaceutically acceptable salts orhydrates of the compounds of Formula XII, may include:

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-m-tolyloxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.1);

2-(5-Methyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-piperidin-1-yl-ethanone(SMC 34.3);

N-Cyclohexyl-2-[5-(naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetamide(SMC 34.4);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.5);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-pyridin-3-yl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.6);

2-(5-Phenoxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-pyrrolidin-1-yl-ethanone(SMC 34.8);

2-(Quinolin-8-yloxymethyl)-oxazole-4-carboxylic acid(tetrahydro-pyran-2-ylmethyl)-amide (SMC 34.10);

2-[4-Phenyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-1-piperidin-1-yl-ethanone(SMC 34.11);

1-Piperidin-1-yl-2-(5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.12);

2-[4-Methyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide(SMC 34.13).

In yet some further particular embodiments, a further example of a SMCmodulator that may be used by the invention, may be the compound

[5-(Naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetic acidmethyl ester (SMC 34.9).

In yet some further particular embodiments, a further example of a SMCmodulator that may be used by the invention, may be the compound

8-(5-Isopropyl-[1,3,4]oxadiazol-2-ylmethoxy)-quinoline (34.2);

In some particular embodiments, specific and non-limiting examples ofthe SMC modulators of the invention, may be pharmaceutically acceptablesalts or hydrates of the compounds of Formula XI, or in some furtherspecific embodiments, pharmaceutically acceptable salts or hydrates ofthe compounds of Formula XIII. More specifically, such compounds mayinclude:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide

(Designated herein as SMC#33); or

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester

(Designated herein as SMC#30).

In some further embodiments, the invention provides an SMC that may beethyl 4-[[3-(4-acetylpiperazin-1-yl)sulfonylbenzoyl]amino]benzoatehaving a structure

(denoted herein as SMC #23)

In yet some other embodiments, the invention provides an SMC that may be10-(3-Chloro-benzyl)-8-(4-methyl-piperazine-1-carbonyl)-5,5-dioxo-5,10-dihydro-5λ6-dibenzo[b,f][1,4]thiazepin-11-onehaving a structure

In yet some alternative particular embodiments of the SMC modulator foruse in accordance with the invention, the compound of Formula XI, mayprovide a compound having the general formula (XIV):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H andstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other may be absent orselected from —(CH₂)—S—, —(CH₂)—, —(CH₂)—O—,

R₃ and R₄ are each independently from each other absent or selected froman aryl or heteroaryl group optionally substituted with at least one ofstraight or branched C₁-C₅ alkyl, halide, nitro and cyano.

In some embodiments, in the SMC modulator for use in accordance with theinvention, R₁ and R₂ are each independently from each other selectedfrom H, methyl and ethyl, at times R₁ and R₂ are each independently fromeach other selected from H and methyl, L1 may be —CH₂—S—, L2 may be—CH₂—O—; R₃ and R₄ are each independently from each other absent orselected from an aryl or heteroaryl group optionally substituted with atleast one of straight or branched C₁-C₅ alkyl, halide, nitro and cyano.

In some further embodiments, in the SMC modulator for use in accordancewith the invention, the R₁ and R₂ may be each independently from eachother selected from H and methyl, L1 is —CH₂—S—, L2 is, —CH₂—O—, R₃ isselected from the group consisting of thiazole, [1,3,4]thiadiazole,[1,3,4]oxadiazole, and R₄ is phenyl.

In yet some alternative particular embodiments of the SMC modulator foruse in accordance with the invention, the compound of Formula I, mayprovide a compound having the general formula (XV):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl and branched C₁-C₁₂ alkyl;

L1 and L2 are each independently from each other selected to be absentor from —(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—,—(CH₂)_(n)—O—, S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n),—(CH₂)_(n)—N—C(═O)—, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—, —NH—(CH₂)_(n)—,—C(═O)—NH—(CH₂)_(n)—; —S—S—(CH₂)_(n)—; —O—(CH₂)_(n)—; —NH—(CH₂)_(n)—;C(═O)—(CH₂)_(n)—; —S—(CH₂)_(n)—; —NH—S(═O)_(n)—(CH₂)_(n)—,—CH₂—S—C(O)—NH—CH₂—, —(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—(NH)_(n)—C(═O)—, —(CH₂)_(n)—N—C(═O)— L1and L2 may be each independently from each other optionally substitutedwith C₁-C₅ alkyl, a ring system containing five to twelve atomsoptionally substituted with C₁-C₅ alkyl

each n, is an integer being independently from each other selected frombe 0 to 5;

R₃ and R₄ are each independently from each other be absent or selectedfrom a ring system containing five to 15 atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), (O)₂, —C(O)—CH₃, —C(O)—O—CH₃, halide, CF₃, nitro, amide, or R₅, R₅is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl or branchedC₁-C₅ alkyl.

In some embodiments, R₁ and R₂ are each independently from each otherselected from H or straight C₁-C₅ alkyl, L1 and L2 are eachindependently from each other selected to be absent or from—(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—, —(CH₂)_(n)—O—,S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n), —(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—, —NH—(CH₂)_(n)—,—C(═O)—NH—(CH₂)_(n)—; —S—S—(CH₂)_(n)—; —O—(CH₂)_(n)—; —NH—(CH₂)_(n)—;C(═O)—(CH₂)_(n)—; —S—(CH₂)_(n)—; —NH—S(═O)_(n)—(CH₂)_(n)—,—CH₂—S—C(O)—NH—CH₂—, —(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—(NH)_(n)—C(═O)—, —(CH₂)_(n)—N—C(═O)— L1and L2 may be each independently from each other optionally substitutedwith C₁-C₅ alkyl, a ring system containing five to twelve atomsoptionally substituted with C₁-C₅ alkyl; each n, is an integer beingindependently from each other selected from be 0 to 5;

R₃ and R₄ are each independently from each other be absent or selectedfrom a ring system containing five to 15 atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), (O)₂, —C(O)—CH₃, —C(O)—O—CH₃, halide, CF₃, nitro, amide, or R₅, R₅is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl or branchedC₁-C₅ alkyl.

In some other embodiments, R₁ and R₂ are each independently from eachother selected from H, straight C₁-C₅ alkyl, L1 is absent and L2 isselected from —(CH₂)—S—, —(CH₂)—O—, —(CH₂)—; R₃ and R₄ are eachindependently from each other be absent or selected from a ring systemcontaining five to 15 atoms, each optionally substituted with at leastone of straight or branched C₁-C₅ alkyl, —C(O)—CH₃, —C(O)—O—CH₃, halide,CF₃, nitro, amide, or R₅, R₅ is an a ring system containing five totwelve atoms optionally substituted by at least one halide or straightC₁-C₅ alkyl or branched C₁-C₅ alkyl.

In some other embodiments, R₁ and R₂ are each independently from eachother selected from H, straight C₁-C₅ alkyl,

L1 is absent and L2 is selected from —(CH₂)—S—, —(CH₂)—O—, —(CH₂)—;

R₃ is a bicyclic ring and R₄ is an aryl or a heteroaryl.

In accordance with some other aspects, the invention relates to at leastone SMC modulator of WASp having the general formula (V):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

each one of X, Y, Z, V, W, T and S may be selected from N, NH and C,

R₆ and R₇ are the same or are different and are independently selectedfrom each other may be L3-R₅,

L3 may be selected from —(CH₂)_(n), —NH—C(O) and C(O)—NH, S(O)₂, C(O), nis an integer between 0 to 5;

R₈ may be selected from a ring system containing five to twelve atoms,each optionally substituted by at least one of straight or branchedC₁-C₅ alkyl, halide, hydroxyl, ester, ether, amide, nitro and hydroxyl,CF₃.

In some embodiments, the invention provides an SMC that may be any oneof:

3-[(3-fluorophenyl)methyl]-5-[1-[2-(trifluoromethyl)phenyl]sulfonylpiperidin-4-yl]-2H-triazolo[4,5-d]pyrimidin-7-one

(Designated herein as SMC #32);

N-[2-(dimethylamino)-2-thiophen-2-ylethyl]-4-[ethyl(phenyl)sulfamoyl]benzamide

(Designated herein as SMC #24);

10-(3-Chloro-benzyl)-8-(4-methyl-piperazine-1-carbonyl)-5,5-dioxo-5,10-dihydro-5λ6-dibenzo[b,f][1,4]thiazepin-11-one(Designated herein as SMC #26);

2-(4-Morpholin-4-yl-6-phenylamino-[1,3,5]triazin-2-ylsulfanyl)-N-(3-nitro-phenyl)-acetamideDesignated herein as SMC #15);

3-(benzylsulfamoyl)-N-[2-(2,6-difluoroanilino)-2-oxoethyl]-N-ethylbenzamide

(Designated herein as SMC #25);

2-[4-[3-(dimethylsulfamoyl)-4-methylphenyl]-1-oxophthalazin-2-yl]-N,N-dimethylacetamide

(Designated herein as SMC #16);

N-(2,5-difluorophenyl)-2-[2-[1-(4-methylphenyl)sulfonylpiperidine-3-carbonyl]hydrazinyl]acetamide

(Designated herein as SMC #21);

2-fluoro-N-[4-[[2-[(4-methyl-5-pyridin-4-yl-1,2,4-triazol-3-yl)sulfanyl]acetyl]amino]phenyl]benzamide

(Designated herein as SMC #31);

2-[2-(naphthalen-2-ylamino)-2-oxoethyl]sulfanyl-N-[(4-propan-2-ylphenyl)methyl]acetamide

(Designated herein as SMC #17)

1-(3-chloro-4-fluorophenyl)-3-[[1-[(2,5-dimethylphenyl)methyl]piperidin-4-yl]methyl]urea

(Designated herein as SMC #28).

It should be appreciated that the invention encompasses any of the SMCsmodulators disclosed herein.

In yet a further aspect, the invention relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone SMC modulator of WASp as described herein. It should be noted thatthe composition of the invention may optionally further comprise atleast one pharmaceutically acceptable carrier/s, excipient/s,auxiliaries, and/or diluent/s.

Some aspects of the invention relate to a pharmaceutical compositioncomprising a therapeutically effective amount of at least one SMCmodulator of WASp having the general formula (I)

or a pharmaceutically acceptable salt, solvate, esters, hydrate,stereoisomer or physiologically functional derivative thereof, or anyvehicle, matrix, nano- or micro-particle comprising the same, saidcomposition optionally further comprises at least one pharmaceuticallyacceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s,wherein

R₁ and R₂ are each independently from each other selected from H,straight or branched C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl,straight or branched C₁-C₁₂ alkoxy, a ring system containing five totwelve atoms, each optionally substituted by at least one of halide,hydroxyl, ester, ether, amide, nitro, —C(═O)—O—(CH₂)_(n)—CH₃, R₅, or—NH—C(═O)—R₅, R₅ is an a ring system containing five to twelve atomsoptionally substituted by at least one halide or straight or branchedC₁-C₅ alkyl;

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to twelve membered saturated or unsaturated ring that mayoptionally include N, O, S, NH, C═N, C═O, S═O, or SO₂ and may beoptionally be substituted with at least one of straight or branchedC₁-C₅ alkyl, hydroxyl, halide and cyano;

L1 and L2 are each independently from each other may be absent orselected from —(CH₂)_(n)—(CH₂—C(O)—N)_(n)—(CH₂)_(n), —(CH₂)_(n)—S—,—(CH₂)_(n)—O—, S(O)₂, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n),—(CH₂)_(n)—N—C(═O)—, S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—S—, NH,—(CH₂)_(n)—S—(CH₂)_(n)—C(O)—NH—(CH₂)_(n)—,—(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—, —NH—(CH₂)_(n)—,—C(═O)—NH—(CH₂)_(n)—; —S—S—(CH₂)_(n)—; —O—(CH₂)_(n)—; —NH—(CH₂)_(n)—;C(═O)—(CH₂)_(n)—; —S—(CH₂)_(n)—; —NH—S(═O)_(n)—(CH₂)_(n)—,—CH₂—S—C(O)—NH—CH₂—, —(CH₂)_(n)—(NH)_(n)—C(═O)—(CH₂)_(n)—N—C(═O)—,S(O)₂—N—(CH₂)_(n), —(CH₂)_(n)—(NH)_(n)—C(═O)—, —(CH₂)_(n)—N—C(═O)— L1and L2 may be are each independently from each other optionallysubstituted with C₁-C₅ alkyl, a ring system containing five to twelveatoms substituted with C₁-C₅ alkyl

each n, is an integer being independently from each other selected frombe 0 to 5;

R₃ and R₄ are each independently from each other be absent or selectedfrom a ring system containing five to 15 atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), (O)₂, —C(O)—CH₃, —C(O)—O—CH₃, halide, CF₃, nitro, amide, or R₅, R₅is an a ring system containing five to twelve atoms optionallysubstituted by at least one halide or straight or branched C₁-C₅ alkyl.

In yet some further aspects thereof, the composition of the inventionmay comprise at least one SMC modulator of WASp degradation having thegeneral formula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other selected to be absentor from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—, —(CH₂)—, —(CH₂)—O—,—NH—(CH₂)—, and each optionally substituted with C₁-C₅ alkyl, a ringsystem containing five to twelve atoms optionally substituted with C₁-C₅alkyl;

R₃ and R₄ are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl.

In some particular embodiments, specific and non-limiting examples ofthe SMC modulators of the compositions of the invention, may bepharmaceutically acceptable salts or hydrates of the compounds ofFormula XI, or in some further specific embodiments, pharmaceuticallyacceptable salts or hydrates of the compounds of Formula XII. Morespecifically, such compounds may include:

1-(2,6-Dimethyl-piperidin-1-yl)-2-[5-(quinolin-8-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-ethanone

(designated herein as SMC 34); or

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-3-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone

(designated herein as SMC 34.7).

In yet some further particular embodiments, specific and non-limitingexamples of the SMC modulators of the compositions of the invention, orpharmaceutically acceptable salts or hydrates of the compounds ofFormula XI, or in some further specific embodiments, pharmaceuticallyacceptable salts or hydrates of the compounds of Formula XII, mayinclude:

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-m-tolyloxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.1);

2-(5-Methyl-[1.3.4]oxadiazol-2-ylsulfanyl)-1-piperidin-1-yl-ethanone(SMC 34.3);

N-Cyclohexyl-2-[5-(naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetamide(SMC 34.4);

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl-ethanone(SMC 34.51;

1-(2,6-Dimethyl-piperidin-1-yl)-2-(5-pyridin-3-yl-[1,3,4]oxadiazol-2-ylsulfanyl)-ethanone(SMC 34.6);

2-(5-Phenoxymethyl-[1,3,4]oxadiazol-2-ylsulfanyl)-1-pyrrolidin-1-yl-ethanone(SMC 34.8);

2-(Quinolin-8-yloxymethyl)-oxazole-4-carboxylic acid(tetrahydro-pyran-2-ylmethyl)-amide (SMC 34.10);

2-[4-Phenyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-1-piperidin-1-yl-ethanone(SMC 34.11);

1-Piperidin-1-yl-2-(5-pyridin-4-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(SMC 34.12);

2-[4-Methyl-5-(quinolin-8-yloxymethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide(SMC 34.13)

In yet some further particular embodiments, a further example of a SMCmodulator that may be used by the invention, may be the compound

[5-(Naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetic acidmethyl ester (SMC 34.9).

In yet some further particular embodiments, a further example of a SMCmodulator that may be used by the invention, may be the compound

[5-(Naphthalen-2-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-acetic acidmethyl ester (SMC 34.9).

8-(5-Isopropyl-[1,3,4]oxadiazol-2-ylmethoxy)-quinoline (34.2)

In some other particular embodiments, specific and non-limiting examplesof the SMC modulators of the compositions of the invention may bepharmaceutically acceptable salts or hydrates of the compounds ofFormula XI, or in some further specific embodiments, pharmaceuticallyacceptable salts or hydrates of the compounds of Formula XIII. Morespecifically, such compounds may include:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide

(Designated herein as SMC#33); or

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester

(Designated herein as SMC#30).

In some embodiments, the pharmaceutical composition comprising atherapeutically effective amount ofN′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamidehaving a structure:

In some other embodiments, the pharmaceutical composition comprising atherapeutically effective amount of1-(2,6-dimethylpiperidin-1-yl)-2-[[5-(quinolin-8-yloxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]ethanonehaving a structure

In some further embodiments, the pharmaceutical composition comprising atherapeutically effective amount is ethyl4-[[3-(4-acetylpiperazin-1-yl)sulfonylbenzoyl]amino]benzoate having astructure

In yet some other embodiments, the pharmaceutical composition comprisinga therapeutically effective amount of10-(3-Chloro-benzyl)-8-(4-methyl-piperazine-1-carbonyl)-5,5-dioxo-5,10-dihydro-5λ6-dibenzo[b,f][1,4]thiazepin-11-onehaving a structure

In accordance with some other aspects, the invention relates to apharmaceutical composition comprising a therapeutically effective amountof at least one SMC modulator of WASp having the general formula (V):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

each one of X, Y, Z, V, W, T and S may be selected from N, NH and C,

R₆ and R₇ are the same or are different and are independently selectedfrom each other may be L3-R₈,

L3 may be selected from —(CH₂)_(n), —NH—C(O) and C(O)—NH, S(O)₂, C(O), nis an integer between 0 to 5;

R₈ may be selected from a ring system containing five to twelve atoms,each optionally substituted by at least one of straight or branchedC₁-C₅ alkyl, halide, hydroxyl, ester, ether, amide, nitro and hydroxyl,CF₃.

In some embodiments, the pharmaceutical composition comprising atherapeutically effective amount of

3-[(3-fluorophenyl)methyl]-5-[1-[2-(trifluoromethyl)phenyl]sulfonylpiperidin-4-yl]-2H-triazolo[4,5-d]pyrimidin-7-one

(Designated herein as SMC #32)

In accordance with some other aspects, the invention relates to apharmaceutical composition comprising a therapeutically effective amountof at least one SMC modulator of WASp having the general formula (IX):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

L4 may be absent or may be selected from —(CH₂)_(n)—, —S—(CH₂)_(n)—,—(CH₂)_(n)—S—;

n is an integer between 0 and 5;

R₁₀ and R₁₁ are each independently from each other absent or selectedfrom a ring system containing five to twelve atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, (═O),(═S), —C(O)—CH₃, —C(O)—O—CH₃, halide, nitro, NH₂.

In some embodiments, L4 is selected from —S—(CH₂)—. In some otherembodiments, R₁₀ and R₁₁ are each independently from each other absentor selected from triazine, piperidine each optionally substituted withat least one NH₂.

In accordance with some other aspects, the invention relates to apharmaceutical composition comprising a therapeutically effective amountof at least one SMC modulator of WASp having the general formula (X):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁₃ and R₁₄ are each independently from each other absent or selectedfrom a ring system containing five to twelve atoms, each optionallysubstituted with at least one of straight or branched C₁-C₅ alkyl, C₁-C₅alkoxy, (═O), (═S), —C(O)—CH₃, —C(O)—O—CH₃, halide, nitro, NH₂,NH—C(O)—CH₃.

In some other embodiments, R₁₀ and R11 are each independently from eachother absent or selected from aryl each optionally substituted with atleast one OCH, NH—C(O)—CH₃.

In some specific embodiments, the pharmaceutical composition maycomprise any of the SMC modulator/s as described by the invention or anyvehicle, matrix, nano- or micro-particle comprising the same.

In yet some further embodiments, the invention provides thepharmaceutical compositions as described herein for use in a method fortreating, preventing, inhibiting, reducing, eliminating, protecting ordelaying the onset of an innate or acquired immune-related disorder orcondition.

In some embodiments, the pharmaceutical composition for use in a methodfor treating, preventing, inhibiting, reducing, eliminating, protectingor delaying the onset of an innate or acquired immune-related disorderor condition, comprising a therapeutically effective amount ofN′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamidehaving a structure:

In some other embodiments, the pharmaceutical composition for use in amethod for treating, preventing, inhibiting, reducing, eliminating,protecting or delaying the onset of an innate or acquired immune-relateddisorder or condition, comprising a therapeutically effective amount of1-(2,6-dimethylpiperidin-1-yl)-2-[[5-(quinolin-8-yloxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]ethanonehaving a structure

In some further embodiments, the pharmaceutical composition for use in amethod for treating, preventing, inhibiting, reducing, eliminating,protecting or delaying the onset of an innate or acquired immune-relateddisorder or condition, comprising a therapeutically effective amount isethyl 4-[[3-(4-acetylpiperazin-1-yl)sulfonylbenzoyl]amino]benzoatehaving a structure

In yet some other embodiments, the pharmaceutical composition for use ina method for treating, preventing, inhibiting, reducing, eliminating,protecting or delaying the onset of an innate or acquired immune-relateddisorder or condition, comprising a therapeutically effective amount of10-(3-Chloro-benzyl)-8-(4-methyl-piperazine-1-carbonyl)-5,5-dioxo-5,10-dihydro-5λ6-dibenzo[b,f][1,4]thiazepin-11-onehaving a structure

It should be appreciated that the small molecule compounds (SMCs) of theinvention are referred to either by their formula or by their numbers,for example, compound #34 may be referred to herein as SMC 34, SMC#34 or#34.

In some specific embodiments, the compositions of the invention may beused for treating hereditary disorder associated with WASp dysfunction.In more specific embodiments, such disorders may be at least one ofWiskott Aldrich Syndrome (WAS) and X-linked thrombocytopenia (XLT), orany condition or disorder associated therewith.

In some specific embodiments, condition or disorder associated with WASand/or XLT, may be also defined as an acquired disorder related to WASpdysfunction. Such conditions or disorders may include but are notlimited to at least one of autoimmune disorders, inflammatory disordersand atopic eczema.

It should be appreciated that in some embodiments, the composition ofthe invention may be used for any immune-related disorders, in someembodiments, any innate or acquired immunodeficiency as detailed inconnection with other aspects of the invention. Specifically, anyprimary (e.g., WAS, XLT and associated conditions) or secondaryimmunodeficiency (e.g., caused by at least one of chemotherapy,radiotherapy, biological therapy, bone marrow transplantation, genetherapy, adoptive cell transfer or any combinations thereof).

It should be appreciated that the SMC modulators used by the methods ofthe invention may be formulated in any vehicle, matrix, nano- ormicro-particle, or composition. Of particular relevance are formulationsof the SMC modulators of the invention adapted for use as a nano- ormicro-particles. Nanoscale drug delivery systems using micellarformulations, liposomes and nanoparticles are emerging technologies forthe rational drug delivery, which offers improved pharmacokineticproperties, controlled and sustained release of drugs and, moreimportantly, lower systemic toxicity. A particularly desired solutionallows for externally triggered release of encapsulated compounds.Externally controlled release can be accomplished if drug deliveryvehicles, such as micelles, liposomes or polyelectrolyte multilayercapsules, incorporate nanoparticle (NP) actuators. More specifically,Controlled drug delivery systems (DDS) have several advantages comparedto the traditional forms of drugs. A drug is transported to the place ofaction, hence, its influence on vital tissues and undesirable sideeffects can be minimized. Accumulation of therapeutic compounds in thetarget site increases and, consequently, the required doses of drugs arelower. This modern form of therapy is especially important when there isa discrepancy between the dose or the concentration of a drug and itstherapeutic results or toxic effects. Cell-specific targeting can beaccomplished by attaching drugs to specially designed carriers.

It should be therefore understood that the invention further encompassesthe use of various nanostructures, including micellar formulations,liposomes, polymers, dendrimers, silicon or carbon materials, polymericnanoparticles and magnetic nanoparticles, as carriers in drug deliverysystems. The term “nanostructure” or “nanoparticle” is used herein todenote any microscopic particle smaller than about 100 nm in diameter.In some other embodiments, the carrier is an organized collection oflipids. When referring to the structure forming lipids, specifically,micellar formulations or liposomes, it is to be understood to mean anybiocompatible lipid that can assemble into an organized collection oflipids (organized structure). In some embodiments, the lipid may benatural, semi-synthetic or fully synthetic lipid, as well aselectrically neutral, negatively or positively charged lipid. In someembodiments, the lipid may be a naturally occurring phospholipid.Examples of lipids forming glycerophospholipids include, without beinglimited thereto, glycerophospholipid. phosphatidylglycerols (PG)including dimyristoyl phosphatidylglycerol (DMPG); phosphatidylcholine(PC), including egg yolk phosphatidylcholine, dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidyl choline(POPC), hydrogenated soy phosphatidylcholine (HSPC),distearoylphosphatidylcholine (DSPC); phosphatidic acid (PA),phosphatidylinositol (PI), phosphatidylserine (PS). Examples of cationiclipids may include, for example, 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP) 1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethyl-ammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);30[N-(N′,N′-dimethylaminoethane) carbamoly]cholesterol (DC-Chol); anddimethyl-dioctadecylammonium (DDAB),N-[2-[[2,5-bis[3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanaminium(DOSPA), and ceramide carbamoyl spermine (CCS), or the neutral lipiddioleoylphosphatidyl ethanolamine (DOPE) derivatized with polylysine toform a cationic lipopolymer.

The lipids may be combined with other lipid compatible substances, suchas, sterols, lipopolymers etc. A lipopolymer may be a lipid modified byinclusion in its polar headgroup a hydrophilic polymer. The polymerheadgroup of a lipopolymer may be preferably water-soluble. In someembodiments, the hydrophilic polymer has a molecular weight equal orabove 750 Da. There are numerous polymers which may be attached tolipids to form such lipopolymers, such as, without being limitedthereto, polyethylene glycol (PEG), polysialic acid, polylactic (alsotermed polylactide), polyglycolic acid (also termed polyglycolide),apolylactic-polyglycolic acid, polyvinyl alcohol, polyvinylpyrrolidone,polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline,polyhydroxypropyloxazoline, polyaspartamide, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide,polyvinylmethylether, polyhydroxyethyl acrylate, derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose. The polymersmay be employed as homopolymers or as block or random copolymers. Thelipids derivatized into lipopolymers may be neutral, negatively charged,as well as positively charged. The most commonly used and commerciallyavailable lipids derivatized into lipopolymers are those based onphosphatidyl ethanolamine (PE), usually,distearoylphosphatidylethanolamine (DSPE).

In some embodiments, the structure forming lipids may be combined withother lipids, such as a sterol. Sterols and in particular cholesterolare known to have an effect on the properties of the lipid's organizedstructure (lipid assembly), and may be used for stabilization, foraffecting surface charge, membrane fluidity.

In some embodiments, a sterol, e.g. cholesterol is employed in order tocontrol fluidity of the lipid structure. The greater the ratiosterol:lipids (the structure forming lipids), the more rigid the lipidstructure is.

Liposomes are often distinguished according to their number of lamellaeand size. The liposomes employed in the context of the presentdisclosure may be multilamellar vesicles (MLVs), multivesicular vesicles(MVVs), small unilamellar vesicles (SUVs), large unilamellar vesicles(LUVs) or large multivesicular vesicles (LMVV).

It should be appreciated that the at least one SMCs of the invention maybe associated with any of the nanostructures described above,specifically, any of the micellar formulations, liposomes, polymers,dendrimers, silicon or carbon materials, polymeric nanoparticles andmagnetic nanoparticles disclosed herein above. The term “association”may be used interchangeably with the term “entrapped”, “attachment”,“linked”, “embedded”, “absorbed” and the like, and contemplates anymanner by which the at least one SMCs of the invention is held. This mayinclude for example, physical or chemical attachment to the carrier.Chemical attachment may be via a linker, such as polyethylene glycol.The association provides capturing of the at least one SMCs of theinvention by the nanostructure such that the release of the at least oneSMCs of the invention may be controllable. Still further, it should beappreciated that in some embodiments, the nanostructure in accordancewith the present disclosure may further comprise at least one targetingmoiety on the surface. Such targeting moiety may facilitate targetingthe SMCs-nanostructures of the invention into a particular target cell,target tissue, target organ or particular cellular organelle target. Thetransporting or targeting moiety may be attached directly or indirectlyvia any linker, and may comprise affinity molecules, for example,antibodies that specifically recognize target antigen on specifichematopoietic cells.

As noted above, the pharmaceutical composition of the invention mayoptionally further comprise at least one pharmaceutically acceptablecarrier/s, excipient/s, auxiliaries, and/or diluent/s. “Pharmaceuticallyor therapeutically acceptable carrier” refers to a carrier medium whichdoes not interfere with the effectiveness of the biological activity ofthe active ingredients. As mentioned herein, the compositions providedby the invention optionally further comprise at least onepharmaceutically acceptable excipient or carrier. As used herein“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except as any conventional media oragent is incompatible with the active ingredient, its use in thetherapeutic composition is contemplated.

As used herein “pharmaceutically acceptable carrier/diluents/excipient”includes any and all solvents, dispersion media, coatings and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcomposition is contemplated.

Pharmaceutical compositions used to treat subjects in need thereofaccording to the invention generally comprise a buffering agent, anagent who adjusts the osmolarity thereof, and optionally, one or morepharmaceutically acceptable carriers, excipients and/or additives asknown in the art. Supplementary active ingredients can also beincorporated into the compositions. The carrier can be solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants.

In various embodiments, the final solution of any of the compositions ofthe invention may be adjusted with a pharmacologically acceptable acid,base or buffer.

The pharmaceutical composition of the invention can be administered anddosed by the methods of the invention, in accordance with good medicalpractice, systemically, for example by parenteral, e.g. intravenous,intraperitoneal or intramuscular injection. In another example, thepharmaceutical composition can be introduced to a site by any suitableroute including intravenous, subcutaneous, transcutaneous, topical,intramuscular, intraarticular, subconjunctival, or mucosal, e.g. oral,intranasal, or intraocular administration.

In some embodiments, the SMCs of the invention as well as pharmaceuticalcompositions thereof may be suitable for systemic administration. Thepharmaceutical composition of the invention can be administered anddosed by the methods of the invention, in accordance with good medicalpractice. More specifically, the compositions used in the methods andkits of the invention, described herein after, may be adapted foradministration by systemic, parenteral, intraperitoneal, transdermal,oral (including buccal or sublingual), rectal, topical (including buccalor sublingual), vaginal, intranasal and any other appropriate routes.Such formulations may be prepared by any method known in the art ofpharmacy, for example by bringing into association the active ingredientwith the carrier(s) or excipient(s).

The phrases “systemic administration”, “administered systemically” asused herein mean the administration of a compound, drug or othermaterial other than directly into the central blood system, such that itenters the patient's system and, thus, is subject to metabolism andother like processes. The phrases “parenteral administration” and“administered parenterally” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

Systemic administration includes parenteral injection by intravenousbolus injection, by intravenous infusion, by sub-cutaneous,intramuscular, intraperitoneal injections or by suppositories, bypatches, or by any other clinically accepted method, including tablets,pills, lozenges, pastilles, capsules, drinkable preparations, ointment,cream, paste, encapsulated gel, patches, boluses, or sprayable aerosolor vapors containing these complexes and combinations thereof, whenapplied in an acceptable carrier. Alternatively, to any pulmonarydelivery as by oral inhalation such as by using liquid nebulizers,aerosol-based metered dose inhalers (MDI's), or dry powder dispersiondevices.

In other embodiments the pharmaceutical composition is adapted fortopical administration. By “topical administration” it is meant that thepharmaceutical composition and the carrier may be adapted to any mode oftopical administration including: epicutaneous, oral, bronchoalveolarlavage, ophtalmic administration, enema, nasal administration,administration to the ear, administration by inhalation.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Pharmaceutical compositions used to treat subjects in need thereofaccording to the invention generally comprise a buffering agent, anagent who adjusts the osmolarity thereof, and optionally, one or morepharmaceutically acceptable carriers, excipients and/or additives asknown in the art. Supplementary active ingredients can also beincorporated into the compositions. The carrier can be solvent ordispersion medium containing, for example, water, ethanol, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants.

In some embodiments the SMCs of the invention and pharmaceuticalcomposition thereof, can be administered either alone or in combinationwith other SMC or with any additional therapeutic agent/s. Combinedcompositions as well as combined treatment regimens are thus encompassedby the invention as will be also detailed in connection with the kit ofthe invention.

More specifically, it should be appreciated that in some embodiments,the SMCs of the invention may be used in a combined therapeutic regimen.In non-limiting embodiments, the SMCs may be combined with at least oneof the chemotherapeutic agents discussed above, and/or optionally withany compound that induces differentiation of hematopoietic progenitorcells (e.g., G-CSF), or any biological therapeutic agent. Thus, in someembodiments, the SMCs of the invention may be added in combination withany of the above treatment regimens. The term “in combination with” suchas when used in reference to a therapeutic regimen, refers toadministration or two or more therapies over the course of a treatmentregimen, where the therapies may be administered together or separately,and, where used in reference to drugs, may be administered in the sameor different formulations, by the same or different routes, and in thesame or different dosage form type.

Alternatively, the SMCs treatment may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the second therapeutic agent and the SMCs are administeredseparately, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the secondagent and the SMCs would still be able to exert an advantageouslycombined effect. In such instances, it is contemplated that one wouldadminister both modalities within about 12-24 hours of each other and,more preferably, within about 6-12 hours of each other, with a delaytime of only about 12 hours being most preferred. In some situations, itmay be desirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

The combined treatment regimen encompassed by the invention may requiredifferent compositions and administration modes for each of thecompounds and as such be facilitated by the provision of the differentcomponents (e.g., the SMCs as well as an additional therapeutic agent)in a kit format.

Thus, a further aspect of the invention relates to a kit comprising:

(a) at least one SMC modulator of WASp having the general formula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof,

wherein

R₁ and R₂ are each independently from each other selected from H,straight C₁-C₁₂ alkyl, a ring system containing five to seven atoms,each optionally substituted by at least one a ring system containingfive to seven atoms optionally substituted by at least one halide orstraight C₁-C₅ alkyl

or

R₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl,

L1 and L2 are each independently from each other selected to be absentor from —CH₂—(CH₂—C(O)—N)—(CH₂)₂, —(CH₂)—S—, —(CH₂)—, —(CH₂)—O—,—NH—(CH₂)—, and each optionally substituted with C₁-C₅ alkyl, a ringsystem containing five to twelve atoms optionally substituted with C₁-C₅alkyl;

R₃ and R₄ are each independently from each other absent or selected froma ring system containing five to 12 atoms, each optionally substitutedwith at least one of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅,R₅ is an a ring system containing five to seven atoms optionallysubstituted by at least one halide or straight C₁-C₅ alkyl; and at leastone of:

(b) at least one chemotherapeutic agent;

(c) at least one biological therapy agent; and

(d) at least one agent that induces differentiation of hematopoieticprogenitor cells.

Still further, in addition to therapies based solely on the delivery ofthe SMCs, combination therapy is specifically contemplated. In thecontext of the present invention, it is contemplated that the SMCstherapy could be used similarly in conjunction with other agentscommonly used for the treatment of neutropenia, leucopenia andthrombocytopenia.

To achieve the appropriate therapeutic outcome, using the methods andcompositions of the present invention, one would generally provide acomposition comprising the SMCs and at least one other therapeutic agent(second therapeutic agent). In the present invention, it is contemplatedthat the second therapeutic agent may involve the administration orinclusion of at least one additional factor that may in some specificembodiments be selected from among EPO, G-CSF, M-GDF, SCF, GM-CSF,M-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, or other various interleukins, IGF-1, LIF,interferon (such as a, beta, gamma or consensus), neurotrophic factors(such as BDNF, NT-3, CTNF or noggin), other multi-potent growth factors(such as, to the extent these are demonstrated to be such multi-potentgrowth factors, flt-3/flk-2 ligand, stem cell proliferation factor, andtotipotent stem cell factor), fibroblast growth factors (such as FGF),and analogs, fusion molecules, or other derivatives of the above.

As noted above, the present invention further encompasses a combinedtreatment with further agents that may induce differentiation ofhematopoietic stem cells. Of particular interest is the G-CSF.Granulocyte-colony stimulating factor (G-CSF), also known ascolony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulatesthe bone marrow to produce granulocytes and stem cells and release theminto the bloodstream. Functionally, it is a cytokine and hormone, a typeof colony-stimulating factor, and is produced by a number of differenttissues. G-CSF also stimulates the survival, proliferation,differentiation, and function of neutrophil precursors and matureneutrophils. The recombinant human G-CSF (rhG-CSF) synthesized in an E.coli expression Filgrastim, was first marketed by Amgen with the brandname Neupogen. Filgrastim (Neupogen) and PEG-filgrastim or lenograstim(Neulasta) are two commercially-available forms of rhG-CSF. Severalbio-generic versions are now also available in markets such as Europeand Australia, and are all applicable for the combined treatment regimenencompassed by the present invention. In oncology and hematology, arecombinant form of G-CSF is used with certain cancer patients toaccelerate recovery and reduce mortality from neutropenia afterchemotherapy, allowing higher-intensity treatment regimens.

According to some embodiments, the kit of the invention may furthercomprise container means for containing the different components of thekit of the invention or any dosage forms thereof. The term “container”as used herein refers to any receptacle capable of holding at least onecomponent of a pharmaceutical composition of the invention. Such acontainer may be any jar, vial or box known to a person skilled in theart and may be made of any material suitable for the componentscontained therein and additionally suitable for short or long termstorage under any kind of temperature. More specifically, the kitincludes container means for containing separate compositions; such as adivided bottle or a divided foil packet however, the separatecompositions may also be contained within a single, undivided container.Typically the kit includes directions for the administration of theseparate components. The kit form is particularly advantageous when theseparate components are preferably administered in different dosageforms (e.g., oral and parenteral), are administered at different dosageintervals, or when titration of the individual components of thecombination is desired by the prescribing physician.

It should be appreciated that the invention further encompasses infurther aspects thereof, any of the nucleic acid constructs describedherein, specifically, any of the mutated WASp constructs described inthe Examples, that encode the different WASp mutants or any fusionproteins thereof. More specifically, any nucleic acid construct thatcomprises the nucleic acid sequence encoding the WASp mutant R86C,specifically, the mutant comprising the amino acid sequence as denotedby SEQ ID NO. 13. In yet some further specific embodiments, theinvention refers to a nucleic acid construct comprising the nucleic acidsequence as denoted by SEQ ID NO. 14, encoding the R86C mutant. In yetsome further embodiments, the invention provides any constructcomprising the nucleic acid sequence encoding the WASp mutant Y107C,specifically, the mutant comprising the amino acid sequence as denotedby SEQ ID NO. 15. In yet some further specific embodiments, theinvention refers to a nucleic acid construct comprising the nucleic acidsequence as denoted by SEQ ID NO. 16, encoding the Y107C mutant. Instill further embodiments, the invention provides any constructcomprising the nucleic acid sequence encoding the WASp mutant A134T,specifically, the mutant comprising the amino acid sequence as denotedby SEQ ID NO. 17. In yet some further specific embodiments, theinvention refers to a nucleic acid construct comprising the nucleic acidsequence as denoted by SEQ ID NO. 18, encoding the A134T mutant. In yetsome further embodiments, the invention further pertains to any cellline or transgenic animal expressing the constructs of the invention.Particular specific embodiments for cell lines provided by the inventionrelate to the WASp knockout Jurkat T-cell lines that further express anyof the mutated WASps of the invention or any derivative or fusionproteins thereof, specifically, WASp knockout Jurkat T-cell lineexpressing the Yellow fluorescent protein (YEP) fusion protein with theWASp mutants. More specifically, WASp knockout Jurkat T-cell lineexpressing the YEP-WASp R86C mutant, in yet some further embodiments theWASp knockout Jurkat T-cell line expressing the YEP-WASp Y107C mutant.Still further, in some embodiments, the invention provides the WASpknockout Jurkat T-cell line expressing the YEP-A134T mutant.

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

The term “about” as used herein indicates values that may deviate up to1%, more specifically 5%, more specifically 10%, more specifically 15%,and in some cases up to 20% higher or lower than the value referred to,the deviation range including integer values, and, if applicable,non-integer values as well, constituting a continuous range. As usedherein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. This termencompasses the terms “consisting of” and “consisting essentially of”.The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.Throughout this specification and the Examples and claims which follow,unless the context requires otherwise, the word “comprise”, andvariations such as “comprises” and “comprising”, will be understood toimply the inclusion of a stated integer or step or group of integers orsteps but not the exclusion of any other integer or step or group ofintegers or steps.

It should be noted that various embodiments of this invention may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range. Whenever a numerical range isindicated herein, it is meant to include any cited numeral (fractionalor integral) within the indicated range. The phrases “ranging/rangesbetween” a first indicate number and a second indicate number and“ranging/ranges from” a first indicate number “to” a second indicatenumber are used herein interchangeably and are meant to include thefirst and second indicated numbers and all the fractional and integralnumerals there between.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub combination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

Other purposes and advantages of the invention will become apparent asthe description proceeds. While in the foregoing description describesin detail only a few specific embodiments of the invention, it will beunderstood by those skilled in the art that the invention is not limitedthereto and that other variations in form and details may be possiblewithout departing from the scope and spirit of the invention hereindisclosed or exceeding the scope of the claims.

The present invention as defined by the claims, the contents of whichare to be read as included within the disclosure of the specification,and will now be described by way of example with reference to theaccompanying Figures.

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

Various embodiments and aspects of the present invention as delineatedherein above and as claimed in the claims section below findexperimental support in the following examples.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, methods steps, and compositionsdisclosed herein as such methods steps and compositions may varysomewhat. It is also to be understood that the terminology used hereinis used for the purpose of describing particular embodiments only andnot intended to be limiting since the scope of the present inventionwill be limited only by the appended claims and equivalents thereof.

EXAMPLES

The following examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Standard molecular biology protocols known in the art not specificallydescribed herein are generally followed essentially as in Sambrook etal., Molecular cloning: A laboratory manual, Cold Springs HarborLaboratory, New-York (1989,1992), and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1988).

Standard organic synthesis protocols known in the art not specificallydescribed herein are generally followed essentially as in Organicsyntheses: Vol. 1-79, editors vary, J. Wiley, New York, (1941-2003);Gewert et al., Organic synthesis workbook, Wiley-VCH, Weinheim (2000);Smith & March, Advanced Organic Chemistry, Wiley-Interscience; 5thedition (2001).

Standard medicinal chemistry methods known in the art not specificallydescribed herein are generally followed essentially as in the series“Comprehensive Medicinal Chemistry”, by various authors and editors,published by Pergamon Press.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Standard molecular biology protocols known in the art not specificallydescribed herein are generally followed essentially as in Vanderkerken KThe 5T2MM murine model of multiple myeloma: maintenance and analysis.[Methods Mol. Med. 113:191-205 (2005); Epstein J. The SCID-hu myelomamodel. Methods Mol. Med. 113:183-90 (2005)].

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in anon-limiting fashion.

Materials and Reagents Antibodies:

The following antibodies were used for imaging: mouse anti-CD3c (UCHT).The following primary antibodies were used for immunoprecipitations andwestern blotting: rabbit anti-Ub (DAKO), mouse anti-GFP (Roche), mouseanti-WASp D1 and rabbit anti-WASp H-250 (Santa Cruz), rabbit anti-WIP(Santa Cruz), rabbit anti-PKCθ (Epitomics), and mouse anti-GAPDH(Biodesign, and Santa Cruz). The following primary antibodies were usedfor flow-cytometry: PE-Cy5™-conjugated mouse anti-human CD69 (Biolegend)and purified anti-activated human LFA-1 antibody from KIM127 hybridoma(Sigma-Aldrich). HRP-conjugated secondary antibodies used include goatanti-mouse (Jackson) and goat anti-rabbit (Jackson). For flow-cytometry,secondary Alexa 488-conjugated goat anti-mouse IgG antibody was used(Jackson).

Reagents:

Small molecule compounds (SMCs) used for the following experiments werepurchased from ChemBridge Corporation, Sigma-Aldrich Corporation andAKos GmbH, were dissolved in DMSO as per the manufacturer's instructionsand diluted gradually in matching cell medium containing 25 mM HEPES forcell incubation.

Pools of three independent RNA duplexes specific for human PKCθ wereobtained from Invitrogen and have the following sequences:GAGUCUCCGUUGGAUGAGGUGGAUA (denoted by SEQ ID NO. 3),GCAUCCGUUUCUGACGCACAUGUUU (denoted by SEQ ID NO. 4) andCCGGCCGAAAGUGAAAUCACCAUUU, (denoted by SEQ ID NO. 5).

Pools of non-targeting (nonspecific) negative control siRNA duplexeswere obtained from Dharmacon and have the following sequences:UAGCGACUAAACACAUCAA (denoted by SEQ ID NO. 6), UAAGGCUAUGAAGAGAUAC(denoted by SEQ ID NO. 7), AUGUAUUGGCCUGUAUUAG (denoted by SEQ ID NO.8), AUGAACGUGAAUUGCUCAA (denoted by SEQ ID NO. 9), andUGGUUUACAUGUCGACUAA (denoted by SEQ ID NO. 10).

Expression Vectors and Plasmid Construction

YFP-WIP, YFP-WASp and CFP-WASp plasmids were obtained as was previouslydescribed (Fried et al, 2014; Reicher et al, 2012). Exogenous plasmidssolely encoding the CDS of WASp. The expression vectors pEYFP-C1,pEYFP-N1, pECFP-C1, and pECFPN1 were obtained from Clontech, andpcDNA3.1+/Hygro was obtained from Invitrogen. Complementary DNA (cDNA)encoding human WASp or WIP were cloned into the expression vectors pECFPor pEYFP to obtain the cyan fluorescent protein (CFP)- or yellowfluorescent protein (YFP)-tagged proteins. Aequorea GFP derivatives wererendered monomeric by the A206K substitution.

YFP tagged WASp plasmid, solely encoding the CDS of WASp was utilized tocreate the common WASp mutants (R86C, Y107C or A134T). Molecular WASpmutants were prepared using the QuikChange II XL site-directedmutagenesis kit (Stratagene).

Establishing T-cell lines exclusively expressing human WAS/XLT WASpmutants tagged to YFP was achieved by using addgene PX458 CRISPR/CAS9plasmid with custom guide RNAs targeting the WAS locus.

Experimental Procedures Adaptation of an Existing PDB Model for VirtualScreening of SMCs

An NMR model of the EVH1 domain of N-WASp (PDB ID: 2IFS) (Peterson(2007), the closest homologue of WASp, was downloaded from RCSB proteindata bank. This model was linked to a fragment of WIP. The model wasrendered suitable for virtual screening by deletion of the WIP fragmentand the linking residues, and additional virtual mutagenesis near theubiquitylation sites using Discovery Studio 3.0 software (Accelrys).Thirty four molecules were selected for further evaluation.

CRISPR/CAS9 Gene Knockdown

CRISPR/CAS9 knockdown of endogenous WASp in T-cells was conductedaccording to published protocol (Ran F A et al. 2013). The vectorpSpCas9 (BB)-2A-GFP (PX458) was purchased from Addgene, plasmid #48138.RNA guides were designed to target an intron/exon junction in the WASgene, in order to eliminate gene silencing of exogenous YFP-WASp. RNAguide sequences aimed for WASp locus were constructed using the onlineCRISPR design tool algorithm (Zhang Lab) and NCBI gene. The guides whichreceived the highest score were selected to be subcloned into thepSpCas9 (BB)-2A-GFP (PX458) vector.

The RNA guides have the following sequences:

aaacTCGCTGGAGATG TAAGTGGATc as denoted by SEQ ID NO. 11 andcaccgATCACTTACATCTCCAGCGA as denoted by SEQ ID NO. 12.

The sequence present in WASp exons is bold. The sequence that iscomplementary to WASp intron (chromosome X, GRCh38.p7 nucleotides1883-1890) is underlined. Knockout of endogenous WASp was conducted inJurkat cells alone or in Jurkat cells stably expressing YFP-WASpcontaining the following mutations, R86C (as denoted by SEQ ID NO. 13and encoded by SEQ ID NO. 14) or Y107C (as denoted by SEQ ID NO. 15 andencoded by SEQ ID NO. 16).

Cell Transfection, Generation of Stable Cells and FACS Analysis

Jurkat E6.1 T cells were transfected with a Lonza Nucleofector™ 2bDevice using the manufacturer's protocol H-10. Stable clones werederived from transiently transfected cells with a combination of drugselection and cell sorting. Cells transiently expressing chimericproteins were selected in Neomycin. Fluorescence analysis and cellsorting were performed using the FACSARIA (Becton Dickinson Biosciences)and FlowJo software (Pauker M H, et al. 2011).

For the establishment of T-cell lines exclusively expressing humanWAS/XLT WASp mutants tagged to YFP, after transfection, cells wereseeded onto a 96 wells plate, diluted to a concentration of 1 cell perwell. After colony growth, individual colonies were screened via westernblot using anti-WASp antibody. Desired colonies were those thatexpressed only exogenous YFP-WASp at a higher molecular weight (92 kDa)and which demonstrated no expression of endogenous WASp (65 kDa).

MicroScale Thermophoresis (MST) Measurements

YFP-tagged WASp was obtained from cell lysates of Human Embryonic Kidney(HEK) 293T, transiently transfected with YFP-WASp/WAVE2/N-WASp byDNA-calcium phosphate co-precipitation as previously described (Reicheret al, 2012). MST measurements were performed using the proteinpurification-free method described by Khavrutskii et al. (Khavrutskii etal, 2013). Briefly, SMCs were serially diluted over three orders ofmagnitude (100 μM-3 nM) in 1×PBS supplemented with 0.05% Tween-20 in 200μL PCR-tubes. Then, YFP-WASp/WAVE2/N-WASp-containing cell lysate wasadded from a doubly diluted sample in the same buffer and the sampleswere gently mixed. The samples were allowed to incubate at roomtemperature for 30 min before being loaded into standard-treatedMonolith™ capillaries (NanoTemper). After loading into the instrument(Monolith NT.115, NanoTemper), the samples were measured by standardprotocols at 60% MST power. The changes of the fluorescentthermophoresis signals were plotted against the concentration of theserially diluted SMCs. K_(D) values were determined using the NanoTemperanalysis software (NT Analysis 1.5.37 or 2.2.31).

Primary PBMC and Platelet Isolation and Stimulation

Human primary PBMCs were isolated from whole blood of healthy donors, aspreviously described (Barda-Saad et al, 2005). The cells were activatedwith anti-CD3ε (OKT3, 10 μg/ml) and anti-CD28 (10 μg/ml) for 30 min onice. The cells were then warmed to 37° C. for 10 min and stimulated withanti-human IgG (50 μg/ml) for 2 min. Human primary platelets wereisolated from thrombocyte-enriched platelet rich plasma peripheral bloodof healthy donors, as previously described by Abcam. Briefly, plateletrich plasma (PRP) was diluted with HEP buffer (140 mM NaCl, 2.7 mM KCl,3.8 mM HEPES, 5 mM EGTA, pH 7.4) at 1:1 ratio (v/v) and centrifuged at100×g for 15 min at room temperature (with no brake applied) to pelletcontaminating red and white blood cells. Platelets were then pelletedthrough plasma by centrifugation at 800×g for 15 min at room temperature(with no brake applied). The washed platelets were resuspended inTyrode's buffer (134 mM NaCl, 12 mM NaHCO₃, 2.9 mM KCl, 0.34 mM Na₂HPO₄,1 mM MgCl₂, 10 mM HEPES, 5 mM glucose, 3 mg/ml BSA, pH 7.4). Plateletsand Meg-01 cells were stimulated with TRAP-6 (10 μM and 100 μM,respectively) at room temperature and at 37° C., respectively, forindicated times.

In Vitro Incubation of Hematopoietic Cells with SMCs

DMSO-soluble stock solutions of SMCs were gradually hydrated with thematching cell medium, containing 25 mM HEPES, by repeated cycles ofintroducing and mixing less than 20% (v/v) hydrous buffer to theDMSO-soluble SMCs. The hydrated SMCs were then added directly to thetarget cells, at final concentration of 40 μM or 100 μM, for indicatedtimes.

Immunoprecipitation and Western Blotting

Immunoprecipitations and western blotting analysis were performed aspreviously described (Reicher et al, 2012). Briefly, platelets (2×10⁸cells per sample) were lysed by the addition of an equal volume of 2×lysis buffer (30 mM HEPES pH7.4, 300 mM NaCl, 2 mM phenylmethyl sulfonylfluoride (PMSF), 2 mM sodium orthovanadate, 2% Triton X-100 and completeprotease inhibitor tablets (Roche)). MEG-01 cells (2×10⁷ cells persample) were lysed in 1.25×lysis buffer. Proteasome activity was blockedby addition of MG132 (AdooQ) to platelets and Meg-01 cell medium atfinal concentration of 10 μM, for 1 h before the cells were harvested.Densitometric analysis of band intensities was performed with ImageJsoftware, with final results normalized with WASp or GAPDH as loadingcontrols for immunoprecipitated samples and whole-cell lysates,respectively. Relative protein abundance or relative extent ofco-precipitated protein was compared to the relevant control.

Extracellular Staining of Lymphocytes for Flow Cytometry

PBMCs or Jurkat cells (about 3×10⁵ cells) were incubated for 4 h, at 37°C. with 50 ng/ml PMA and 250 ng/ml lonomycin, or 40 ng/ml PMA and 6 μMIonomycin, respectively, or left untreated. After incubation, the cellswere collected and stained with 1 μg/ml anti-active human LFA-1 antibody(purified from KIM127 hybridoma sup, ATCC Cat#: CRL-2838, Sigma) for 30min in 37° C., followed by staining with Alexa488-Fluor goat anti-mouseIgG1 secondary antibody (Molecular Probes, Cat#: A-21121), 30 min onice. Cells were then co-stained with PE-Cy5™-conjugated mouse anti-humanCD69 (BD Pharmingen, Cat#: 555532) to measure lymphocyte activation.

Cell Proliferation Assay

Lymphocyte proliferation was assessed using an XTT-based CellProliferation Kit (Biological Industries Ltd.) according to themanufacturer's instructions.

Lymphocyte Migration Assay

Approximately 1×10⁵ lymphocytes were seeded over chambered coverslips(LabTek) pre-coated with 6 μg/ml ICAM-1 and 2 μg/ml SDF-1α. Cells wereincubated at 37° C., 5% CO₂ and DIC images were acquired with the ZeissObserver Z1 inverted microscope every 5 s for 20 min under a 20×objective lens using Zen software. The random movement (Displacement andvelocity) of the cells was automatically tracked and analyzed usingTrackMate plugin of ImageJ software. An individual trace was assigned toeach of the analyzed cells.

Spreading Assay

Spreading assays were performed as previously described (Fried et al,2014; Barda-Saad 2005). Briefly, T cells (2×10⁶ cells/ml) were seeded onthe bottom of chambered cover glasses (LabTek) that were pre-coated withanti-CD3 stimulatory monoclonal antibodies (10 μg/ml). The cells wereincubated in imaging buffer [RPMI without phenol red containing 10%fetal calf serum (FCS) and 25 mM HEPES] at 37° C., 5% CO₂, for the timesindicated in the figure legends. Cells were fixed for 25 min with 4%paraformaldehyde in phosphate-buffered saline (PBS) and then were washedthree times with PBS.

Measurement of Platelet Intra-Cellular Calcium Concentration

Cells were incubated with 5 μM Indo-1-acetoxymethylester (Indo-1-AM,Teflabs) in Tyrode's buffer at 37° C. for 20 min. The cells were washedonce, resuspended in Tyrode's buffer containing 10 mM HEPES andmaintained at room temperature for 20 min. The cells were incubated at37° C. for 5 min before measurements and then stimulated with 10 μMTRAP-6. The Ca²⁺ influx was measured by spectrofluorometer using theSynergy 4 Microplate Reader (Bio Tek).

FRET Correction and Calculation

Double-color FRET was performed as described previously (Barda-Saad etal, 2005; Pauker, 2011, Pauker, 2012). The inventors used CFP(excitation wavelength: 468 nm; emission filter wavelength: 465 to 510nm) as a donor, and YFP (excitation wavelength: 514 nm; emission filterwavelength: 530 nm long-pass, LP) as an acceptor. The non-FRETcomponents were calculated and removed with calibration curves derivedfrom images of single-labeled cells containing CFP or YFP, as previouslydescribed (Barda-Saad et al, 2005). Sets of reference images wereobtained with the same acquisition parameters as those used for the FRETexperimental images. Bleed-through components were calculated as afunction of the intensity of the expressed fluorescent proteins withdata gathered from single-labeled cell lines. By using the intensitymeasured pixel by pixel through the different filter sets, as well ascross-talk elements that were isolated from the control, single-labeledcells, the measured FRET was corrected and the actual FRET efficiency atevery pixel was determined. The relative FRET efficiency (FRETeff) wascalculated on a pixel-by-pixel basis with the following equation:FRETeff=FRETcorr/(FRETcorr+donor)×100%, where FRETcorr is the pixelintensity in the corrected FRET image, and donor is the intensity of thecorresponding pixel in the appropriate donor channel image. To increasethe reliability of the calculations and to prevent low-level noise fromdistorting the calculated ratio, the inventors excluded pixels below 50intensity units, as well as saturated pixels from the calculations, andset their intensities to zero. These pixels are shown in black in thepseudo-colored FRET efficiency images. To estimate the reliability ofthe obtained FRET efficiency values and to exclude the possibility ofobtaining false-positive FRET, the inventors prepared cells expressingfree CFP and free YFP as negative controls. The FRET efficiency in thenegative control system was measured and calculated in the same manneras that in the main experiment. FRET efficiency values obtained from thenegative control samples were subtracted from the values obtained in themain experiments.

Example 1 Virtual Screening Enables the Identification of WASp-BindingSMCs

In order to find promising SMCs candidates capable of preventing WASpubiquitylation-dependent degradation, an NMR model of a recombinant EVH1domain of N-WASp fused to a short WIP peptide (PDB ID: 2IFS) was adapted(FIG. 1) (Peterson, F C et al. 2007). The WIP and the linker fragmentswas removed, and in silico mutagenesis was performed in order tosimulate a WASp model. Three-dimensional structures of SMCs wereconstructed from chemical formulae using the FAST algorithm. UsingPatchDock molecular docking software (D. Schneidman-Duhovny, et al,2005), models of putative SMC-WASp binding structures were generated,and were filtered taking only the highest scoring model for each SMC 3Dstructure-WASp pair. These models were then further filtered bydiscarding any structure depicting a binding not in close proximity toWASp degradation site (defined as more than 5 Å between the SMC andeither of the lysine residues 76 or 81). Finally, the remaining modelswere scored by a machine learning trained classifier as illustrated inFIG. 2).

Following this in silico screening, the inventors identified eight SMCs(#6, #23, #24, #25, #30, #32, #33, #34; see Table 1). The inventorsvalidated their binding to human WASp, using MST, as described inExperimental procedures. As demonstrated in FIG. 3, four of these SMCs(#6, #30, #33, #34) showed binding to WASp, with dissociation constants(K_(D)) that ranged between 25 nM to 0.79 μM (FIG. 3A-3D), while anirrelevant SMC, Y-27632 (Rho-associated protein kinase inhibitor) didnot show binding to WASp (FIG. 3E). Strikingly, no binding wasdemonstrated between WASp-binding SMC #34 to the ubiquitously expressedWASp paralogs, i.e. N-WASp (FIG. 3F) and WAVE2 (FIG. 3G), indicating itsspecificity and precluding off-target effects. The SMCs thatspecifically bind the degradation pocket of WASp were further evaluatedby the inventors.

TABLE 1 The SMC molecules Molecule IUPAC name SMC #6N-[(2R,4R,6S)-2-(4-chlorophenyl)-6-(1-methylbenzotriazol-5-yl)oxan-4-yl]acetamide SMC #23 ethyl4-[[3-(4-acetylpiperazin-1- yl)sulfonylbenzoyl]amino]benzoate SMC #24N-[2-(dimethylamino)-2-thiophen-2-ylethyl]-4-[ethyl(phenyl)sulfamoyl]benzamide SMC #253-(benzylsulfamoyl)-N-[2-(2,6-difluoroanilino)-2-oxoethyl]-N-ethylbenzamide SMC #30 Methyl3-[[4-(cyclopentylcarbamoyl)phenyl]methyl]-4-oxo-2-sulfanylidene-1H-quinazoline-7-carboxylate SMC #323-[(3-fluorophenyl)methyl]-5-[1-[2-(trifluoromethyl)phenyl]sulfonylpiperidin-4-yl]-2H-triazolo[4,5-d]pyrimidin-7-one SMC #33N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide SMC #341-(2,6-dimethylpiperidin-1-yl)-2-[[5-(quinolin-8-yloxymethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]ethanone

Example 2 WASp-Binding SMCs Upregulate WASp Expression

Having identified SMCs that were experimentally validated to bind WASpin the proximity of WASp degradation site, the inventors examinedwhether these SMCs can increase WASp expression in hematopoietic cells.Platelets from healthy donors were pre-incubated with SMC #6 (40 μM) ornon-treated control (DMSO) for 15 min followed by stimulation andwestern blot analysis. As shown in FIG. 4A and illustrated in thesummarizing graph of FIG. 4B, stimulation of non-treated controlplatelets consistently resulted in downregulation of WASp, confirmingprevious observations that WASp in platelets is degraded followingplatelet activation (Lutskiy et al, 2007, Shcherbina et al, 2001). Morespecifically, incubation with SMC #6 attenuated WASp degradation inactivated platelets, compared with control platelets, with prominenteffect observed following 15 min of incubation (FIG. 4A, 4B). Theseresults demonstrate a WASp-upregulating activity in active plateletsduring 15 min of incubation, which is facilitated by SMC #6.

In freshly isolated peripheral blood mononuclear cells (PBMCs),treatment with SMC#6 also attenuated WASp degradation after 15 min ofincubation, and this effect was more pronounced after 2 h (FIG. 4C) andillustrated in the summarizing graph of FIG. 4D. These resultsdemonstrate an optimal WASp-upregulating activity, which is facilitatedby SMC #6 during 15 min or 2 h of incubation, in active platelets andPBMCs, respectively.

Encouraged by these results, the inventors next examined the effect ofthe other WASp-binding SMCs on WASp expression in activated JurkatT-cell line, PBMCs and platelets that is illustrated in FIG. 5.Strikingly, under these conditions, treatment with each of the indicatedSMCs resulted in WASp upregulation, up to 4.5, 3.3, 2-fold, comparing tonon-treated platelets, PBMCs or T-cells, respectively (FIG. 5A-5D).Similar results were shown in FIG. 6A-6D for further SMCs, as indicatedtherein. Therefore, targeting the degradation pocket of WASp by the SMCsof the invention provides protection and may therefore restore WASpfunctions.

Example 3 WASp Undergoes Ubiquitylation-Dependent ProteasomalDegradation in Megakaryocytes and Platelets

Insufficient WASp expression in platelets and megakaryocytes is thecause of microthrombocytopenia of WAS/XLT patients (Albert et al 2010,Lutskiy et al, 2007). It is established that WASp is degraded inplatelets and megakaryocytes following their activation (Lutskiy et al,2007, Shcherbina et al, 2001). However, it is unclear whether thisdownregulation mechanism is executed via the ubiquitin-proteasomepathway, as previously demonstrated for lymphocytes (Fried et al, 2014;Reicher et al, 2012). Because the mechanism of action of WASp-bindingSMCs is via blocking the ubiquitylation pathway of WASp, the inventorshypothesized that this degradation pathway may be relevant toplatelets/megakaryocytes as well. The inventors previously demonstratedthat in lymphocytes PKCθ associates with the WIP-WASp complex early inthe activation process, and later, phosphorylation of WIP by PKCθresults in WIP-WASp slight dissociation and subsequent WASpubiquitylation (Fried et al, 2014). To examine whether similar pathwaysexist in platelets, the inventors stimulated freshly-isolated plateletsfrom healthy donor peripheral blood, lysed the platelets 30 sec, 2 minand 15 min post-stimulation and analyzed the interactions between WIP,WASp and PKCθ using co-immunoprecipitation analysis. As presented inFIG. 7A, both WASp and PKCθ co-precipitated with WIP after 30 sec and 2min of activation, and this interaction was substantially decreasedafter 15 min of activation. Similarly, reciprocal co-precipitation wasdetected between WASp and WIP during the first two minutes of activationwhen WASp was immunoprecipitated (FIG. 7B). The inventors then testedwhether this triplet interaction in platelets leads to WASpubiquitylation. Following activation, WASp was immunoprecipitated fromthe lysates of freshly-isolated platelets, and the lysates were probedby western blot for ubiquitin and WASp. WASp ubiquitylation was detectedas a ladder of bands above 65 kDa, with a prominent band at ˜81 kDa,representing ubiquitylated WASp (Reicher et al, 2012) (FIG. 7C). Thisubiquitylation was more intense after 2 min and 15 min of activation. Inorder to examine whether WASp ubiquitylation in platelets marks it forproteasomal degradation, WASp expression was analyzed in platelets after15 min of activation, with or without the addition of MG132 proteasomeinhibitor. Western blot analysis revealed more than 2-fold increase inWASp expression when the platelets were treated with the proteasomeinhibitor (FIG. 7D), suggesting that in platelets WASp is downregulatedin the ubiquitin-proteasome pathway. As megakaryocytes may also be afuture target for WASp-protecting SMCs, the inventors next examinedwhether ubiquitylation-mediated proteasomal degradation of WASp occursalso in megakaryocytes. Therefore, Meg-01 cells were either treated withMG132 or left untreated, lysed following stimulation and the lysateswere immunoprecipitated with anti-WASp antibody. As shown in FIG. 7E,probing immunoprecipitates of WASp with anti-ubiquitin and anti-WASpantibodies revealed WASp ubiquitylation pattern similar to that oflymphocytes (Fried et al, 2014; Reicher et al, 2012) and platelets. WASpubiquitylation occurred following activation and was more intense incells that were treated with MG132, suggesting that WASp enters theproteasomal pathway following its ubiquitylation in megakaryocytes.Accordingly, more WASp was detected in the whole cell lysates (W.C.L) ofMeg-01 cells that were treated with MG132 (FIG. 7G). The possibleinvolvement of PKCθ in WASp degradation in megakaryocytes was nextexamined in stimulated Meg-01 cells that were either transfected withPKCθ-specific siRNA or non-specific scrambled siRNA. As shown in FIG.7F, PKCθ siRNA achieved 70% silencing of PKCθ in the treated cells,which in turn resulted in 1.35-fold increase of WASp expression in thetreated megakaryocytes. Taken together, these data suggest aubiquitylation-dependent degradation mechanism of WASp in platelets andmegakaryocytes, which is similar to that of lymphocytes.

Example 4 WASp-Binding SMCs Effectively Upregulate Cellular Function ofLymphocytes and Platelets

WASp plays a key role in various functions of hematopoietic cells,including cellular activation, proliferation and migration. Thesecellular functions are impaired in WASp-deficient mice and WAS/XLTpatients (Matalon et al 2013). Without being bound by any theory, theinventors hypothesized that upregulating WASp expression by SMCs maypotentially restore normal immune function. Accordingly, upregulation ofWASp expression by SMCs in normal immune cells is expected to enhancecellular function of these cells. To test this hypothesis, the inventorsset up an array of in vitro biological assays, in which several cellularfunctions of lymphocytes and platelets were examined, followingincubation with WASp-binding SMCs.

Lymphocyte activation is a hallmark of a normal immune response. To testwhether treatment with WASp-binding SMCs could upregulate lymphocyteactivation, freshly-isolated PBMCs were incubated with SMCs or control(solvent only) and stimulated with PMA and lonomycin. The cells werethen stained with two different antibodies, which recognize twolymphocyte activation markers, CD69 and an epitope exposed at the activeconformation of the integrin LFA-1 (KIM127). Both were detected byflow-cytometry. In comparison to control cells, treatment with SMC #6,#30, #33 and #34 led to an upregulation of lymphocyte activation, asdetected by FACS (FIG. 8A-8D and FIG. 9C).

Once activated, lymphocytes proliferate to effectively amplify theimmune response. To examine the possible effect of WASp-protecting SMCson the proliferation of lymphocytes, T cell line (Jurkat) or freshlyisolated PBMCs were treated with SMCs and assayed for theirproliferation, using an XTT-based assay. In accordance with theupregulation of lymphocyte activation, treatment with each of theindicated SMCs enhanced cellular proliferation of the treated cells, upto 2-fold, compared to the non-treated cells (FIG. 9D-9E). In line withthese findings, FACS analysis shown in FIG. 9 reveals an upregulation ofJurkat T cells (FIG. 9A, 9B) and PBMCs (FIG. 9C) activation incubatedwith SMC #6.

A common theme of active lymphocytes is their high migratory ability.This enables them to move within tissues and reach and engage targetcells (Burkhardt et al, 2008). Because WASp plays a role in cellularmotility, the ability of WASp-binding SMCs to effectively enhance themigration of lymphocytes was tested. The random movement of SMC-treatedor control cells was automatically tracked and analyzed using livemicroscopy, as described in Experimental procedures. As observed,measured and presented by FIG. 10A-10C, treatment with SMC #6, #30, #33and #34 resulted in a significant 1.7- to 2.83-fold increase in themigration of these cells in comparison to the DMSO-treated cells.Therefore, specific upregulation of WASp expression with the SMCsefficiently enhanced the migration of T lymphocytes.

Unlike lymphocytes, platelets, which are non-nuclear cells, do notundergo proliferation following their activation. However, followingreceptor-mediated stimulation, platelets dramatically elevate theirintra-cellular calcium concentrations, which in turn serve as signalingcues that evoke platelet effector functions, including plateletspreading, adhesion and aggregation (Varga-Szabo et al, 2009).Therefore, the ability of WASp-protecting SMCs to upregulateintra-cellular calcium accumulation in activated platelets was nexttested. Freshly-isolated human platelets were incubated with SMC #6,#30, #33 and #34 or with the solvent (negative control). Followingincubation, platelets were stimulated with thrombin receptor activatorpeptide TRAP-6 (10 μM) and calcium levels were measured byspectrofluorometer, using Indo-1-AM-based calcium assay as described inExperimental procedures. FIG. 11A-11D shows that in accordance with theWASp-upregulating activity of SMCs in lymphocytes, treatment with any ofthe tested SMCs dramatically enhanced intra-cellular calciumconcentration in activated platelets (FIG. 11A-11D). These resultsclearly demonstrate the potential therapeutic effect of WASp-protectingSMCs in platelets.

Example 5 WASp Protection by WASp-Binding SMCs is Achieved ViaAttenuation of WASp Ubiquitylation

WASp-protecting SMCs were screened and selected according to theirpredicted ability to bind and block the degradation sites of WASp. Inorder to validate that this indeed the mechanism of action by whichthese SMCs work, it was examined whether treatment with WASp-bindingSMCs affect the ubiquitylation of WASp in activated platelets andlymphocytes. Freshly-isolated activated or non-activated platelets werepreincubated for 15 min with SMC #6, and #33 or with the solvent (neg.control), and lysed. WASp was then immunoprecipitated from the lysates,and the co-immunoprecipitation of ubiquitin and WASp was assayed bywestern blot. WASp ubiquitylation in active platelets, treated withWASp-binding SMCs was considerably decreased in comparison to activateduntreated cells (FIG. 12A-12B). Similarly, treatment of PBMCs withWASp-binding SMC #6 and #33 downregulated the ubiquitylation of WASp intreated active lymphocytes in comparison to activated negative controlcells (FIG. 12C-12D).

Additional mechanism by which WASp-binding SMCs could protect WASp fromubiquitylation is to strengthen the WIP-WASp interaction, as WIPprotects WASp from proteasomal degradation (Fried et al, 2014; Reicheret al, 2012). To monitor the WIP-WASp interaction the inventors nextemployed the Förster resonance energy transfer, also known asfluorescence resonance energy transfer technology or FRET, which iswidely used to detect molecular interactions in the nanometer resolution(Fried et al, 2014; Barda-Saad et al 2005). The wild type human WASp andWIP were fluorescently tagged with cyan fluorescent protein (CFP) andyellow fluorescent protein (YFP), respectively. T-cells stablyexpressing CFP-WASp and YFP-WIP were treated with SMC #6, #30, #33 and#34 or with the negative control. The cells were plated on stimulatorycoverslips (pre-coated with an anti-CD3 antibody), and then were fixedafter 5 min (FIG. 13A is a confocal micro image and FIG. 13B representsthe percentage of FRET efficiency). Under these conditions, low FRETefficiency was detected in activated T cells that were treated with thesolvent (9.9±1.5, FIG. 13), indicating a very weak interaction betweenWIP and WASp following cellular activation, as expected and previouslypublished (Fried S, et al. 2014). Interestingly, SMC-treated cellsdisplayed very high average FRET efficiency (SMC#6: 22.7±1.4%, SMC#30:24.4±1.4%, SMC#33: 21.4±1.4%, SMC#34: 23.8±1.1%), typical of a stableWIP-WASp interaction, which is usually observed in non-activated cells(FIG. 13). This unexpected result indicates that WASp-binding SMCs coulddirectly or indirectly stabilize the WIP-WASp interaction.

Example 6 WASp-Binding SMCs Restore WASp Expression in LymphocytesExpressing Common Human WAS/XLT Mutations

Having established the effect of WASp-binding SMCs in the upregulationof WASp expression and cellular function in T lymphocytes and platelets,the inventors explored whether these SMCs could restore WASp expressionof known human common WASp mutants that cause to WAS/XLTimmunodeficiencies. For this purpose, Jurkat T-cells were initiallygenerated stably expressing YFP-WASp wt or common WASp mutants includingWASp R86C, WASp Y107C and WASp A134T, as denoted by the amino acidsequences of SEQ ID NO. 13, 14 and 15, respectively, and encoded by thenucleic acid sequence as denoted by SEQ ID NO. 14, 16 AND 18,respectively. As expected, exogenous WASp mutants (R86C, Y107C, A134T)demonstrated lower expression than exogenous WASp wt due to the factthat these mutations have a deleterious impact on WASp stabilization(FIG. 14A). Lymphocytes stably expressing the YFP-WASp mutants were thenincubated with SMCs or control, stimulated and analyzed by western blot.Treatment with WASp-binding SMCs increased WASp expression compared tocontrol-treated cells in YFP-WASp mutant expressing cells (FIG. 14B).

In order to study the effect of WASp-binding SMCs on the prevention ofWASp degradation, it is necessary to establish a physiologicallyrelevant model which accurately resembles the phenotypic characteristicsof defective WASp expression. A limitation to this approach is thatselected SMCs cannot be tested in vivo due to the lack of a valid animalmodel. The only available animal model for studying WAS is the WASknockout mouse, which is completely devoid of the WAS gene and as such,is not a suitable model for testing post translational pathways (SnapperS B, et al. 1998). Hence, generating WASp knockout human cell lines,solely expressing exogenous WASp mutants can serve as invaluable toolsto test the efficacy of SMC mediated therapy. These lines would have thebenefit of exclusively expressing tagged mutant WASp, potentiallyserving as ideal templates to study SMC stabilizing effects withoutendogenous WASp background interference.

To this end, human WASp knockout Jurkat T-cell lines were generatedwhich have no expression of endogenous WASp wt (WAS^(−/−)) (FIG. 14C)and solely express exogenous WASp harboring the common human WAS and XLTmutations, R86C (WAS^(−/−)/WASp R86C) or Y107C (WAS^(−/−)/WASp Y107C),utilizing the CRISPR/CAS9 genome editing system, an emerging powerfultool for custom genomic modification including genomic deletions andpoint mutations (Doudna J A, et al. 2014).

Cells exclusively expressing the YFP-WASp mutants were incubated withSMC#34 or solvent, stimulated and analyzed by western blot. Treatmentwith WASp-binding SMC increased WASp expression compared tocontrol-treated cells (FIG. 14D). These results demonstrate thatWASp-binding SMCs can successfully restore WASp levels in WAS/XLT mutantcells, thus may become a novel therapeutic approach to treat thesediseases.

Finally, the inventors examined the ability of WASp-binding SMC torestore the cellular function of cells that completely lack WASpexpression (WAS^(−/−)) or to upregulate the cellular function of cellsthat exclusively express WASp mutant forms and are deficient toendogenous WASp.

In comparison to control treated cells, treatment with SMC#34 led to anupregulation of lymphocyte activation in cells exclusively expressingYFP-WASp R86C and restored the activation of WAS^(−/−) cells (FIG. 14E).Furthermore, treatment with SMC#34 significantly increased the migrationof WAS^(−/−)/WASp R86C and WAS^(−/−)/WASp Y107C cells and completelyrestored the migration of WAS^(−/−) cells (FIG. 15A-15C).

Collectively, these results clearly demonstrate the mechanism of actionand therapeutic potential of WASp-binding SMCs. In normal restinglymphocytes, megakaryocytes and platelets, WASp is protected fromubiquitylation and degradation via its interaction with WIP. Followingcellular activation, this interaction is partially released, WASpdegradation sites are uncovered which leads to proteasomal degradation.Alternatively, in WAS/XLT patients the WIP-WASp interaction iscompromised due to mutations in WASp N′-terminus. In the absence of, orcomprised WIP protection, WASp is constitutively ubiquitylated anddegraded. The addition of carefully selected SMCs, which bind in a closeproximity to WASp degradation pocket, blocks the exposed degradationsites of WASp, protecting it from ubiquitylation and degradation asillustrated in FIG. 16. Thus, WASp-binding SMCs protect WASp fromdegradation and can upregulate the expression and normal function ofWASp in the hematopoietic cells of WAS/XLT patients.

Example 7 Treating Immunosuppression Caused by Chemotherapy andRadiotherapy The Effect of WASp-Stabilizing SMC in Improving LeukocyteProliferation and Hematopoietic Cell Reconstitution.

WASp was shown to promote lymphocyte growth and survival in both miceand humans. Proliferation of T or B lymphocytes in response tostimulation by anti-T cell antigen receptor (TCR) or anti-IgM,respectively, are impaired in WASp-deficient lymphocytes. Restoration ofWASp expression has been shown to significantly mitigate these defects(Charrier S. et al. Gene therapy 2007). Reduced leukocyte number is acommon side effect of chemotherapy and radiotherapy, which exposescancer patients to life threatening infections. Increasing WASpexpression enables to expedite hematopoietic rejuvenation followingchemo- and radiotherapies by enhancing lymphocyte growth and survival.

To determine the ability of the WASp-stabilizing SMC to improvelymphocyte proliferation, primary hematopoietic cells including T, B,and NK cells are isolated from peripheral blood of healthy donors, usingmagnetic cell separation kits (commercially available EasySep,StemCell). Freshly isolated cells are incubated with theWASp-stabilizing SMCs of the invention and compared to control-SMC. Thecells are then activated with anti-CD3 for T cells, anti-IgM for Bcells, and anti-CD16 for NK cells. Unstimulated cells are examined as acontrol. Cellular proliferation are determined using XTT proliferationassay or by a FACS-based CFSE staining assay, which measuresproliferation by dilution of the fluorescent signal upon cellulardivisions. A significant increase in lymphocyte proliferation uponWASp-stabilizing SMC treatment is expected.

To assess whether the WASp-stabilizing-SMCs of the invention couldrestore normal lymphoid cell counts, mice are subjected to chemotherapyor radiotherapy and hematopoietic recovery is monitored. To determinethe effect of SMC treatment on the immune response followingchemotherapy, C57BL/6 mice are treated with the chemotherapeutic agentsoxaliplatin (5 mg/kg), or doxorubicin (2 mM in 100 ml PBS) injectedintraperitoneally (IP).

For radiotherapy experiments, C57BL/6 mice are briefly anesthetizedusing isoflurane, and subjected to total body X-irradiation at a dose of7.5 Gray (Gy). Mice are used at 6 to 20 weeks of age. The mice areinjected intravenously (IV) with the WASp-stabilizing SMCs of theinvention vs. control and monitored by complete blood cell counts todetermine the absolute numbers of T, B, and NK cells. Blood sample arewithdrawn from the tail vein and the cells are counted primarily using ablood analyzer (KX21N, Sysmex, Kobe, Japan) followed by Giemsa-stainingof blood smears.

Additionally, total cell counts in bone marrow, thymus, and spleen areevaluated after preparation of single-cell suspensions and eliminationof red blood cells.

To confirm normal reconstitution of the hematopoietic system, ascompared of proliferation of specific populations at the expense ofothers, the relative distribution of white blood cell subsets isanalyzed by FACS. Specifically, the following subsets is analyzed:SSClowCD11b-CD90+(T cells), SSClowCD11b− B220+ (B cells), SSClowCD11b(monocytes), SSChighCD11b+ (granulocytes). Although WASp-stabilizingSMCs are not expected to affect monocyte and granulocyte numbers butrather their functions, these cells are analyzed to determine thedistribution of lymphocytes relatively to the entire hematopoietic cellpopulation. Moreover, due to the specific effect of WASp on lymphocyteproliferation and survival, the number and relative distribution of T(CD3+), NK (CD3-CD56+) and B (CD19+) cells is specifically determinedusing FACS. Focusing on these markers enables to determine thedistribution of the major hematopoietic populations. The absolute bloodcounts of these cells is calculated by multiplying the relativepercentages by the total white blood cell counts.

The Use of WASp-Stabilizing SMC in Enhancing Leukocyte Migration andHoming.

Aside from increasing cellular proliferation, which results in theexpansion of specific leukocyte populations, secondaryimmuno-deficiencies can also be treated by boosting the activity andeffector function of mature leukocytes within existing populations. Theefficacy of an immune response is dependent on the timely recruitment ofimmune cells to sites of inflammation. A hallmark of a suppressed immunesystem, as evident in WAS patients, is defective migration of immunecells to inflammation sites. Enhancement of cellular migration improvesthe effectiveness of the immune response. Increased cellular functione.g. migration might compensate for reduced number of cells.

Granulocytes form the host's first line of defense against invadingpathogens with neutrophils being among the first cells to respond.Impaired recruitment of neutrophils to inflammatory sites allows moretime for pathogens to replicate and induce damage. Migration ofneutrophils is impaired in WAS KO mice both in vitro and in vivo(Snapper S. B et al. Journal of leukocyte biology 2005). Thus, improvingneutrophil migration is expected to improve clinical outcomes followinginfections. First, the effects of WASp-stabilizing SMC on neutrophilmigration is examined, without prior chemo- or radiotherapy treatments.Neutrophils are isolated from blood of healthy human donors usingmagnetic bead isolation kit (StemCell), and the cells are incubated withWASp-stabilizing SMC vs. control. Migration is examined using previouslydescribed protocols in response to complement component 5a (c5a), aknown recruiter of phagocytes (Zhang, H et al. Immunity 2006).

Defects in adhesion and migration of neutrophils in WAS appear to bemore apparent under conditions of physiologic shear flow, during whichintegrin attachment is critical. To further examine the effect of WASpupregulation on neutrophil recruitment, the effect of SMC treatment onthe adhesion and migration of neutrophils under conditions ofphysiological shear flow is monitored. Briefly, neutrophils isolatedfrom healthy donors, are treated with WASp-stabilizing SMC, andsubjected to shear flow assay, as previously described (Zhang, H et al.Immunity 2006). Upregulated adhesion and migration of neutrophils isanticipated following treatment with WASp-stabilizing SMC.

Migration of B and T lymphocytes is crucial for their function.Defective migration of T cells causes reduced or delayed homing ofeffector T cells to the inflammatory site, hampering their ability tocorrectly regulate immune function at the infection site, as well astheir ability to carry out direct effector functions. Defective B cellmigration and chemotaxis disrupts the humoral immune response. T and Blymphocytes of WAS patients and WAS KO mice are impaired in theirmigratory capacity (Snapper S. B et al. Journal of leukocyte biology2005). WASp-deficient B cells fail to migrate towards CXCL1359, acrucial chemokine for attracting B cells to the lymphoid follicles, andT cells do not respond to CCL19 and CCL2154, which mediate T cell homingto the secondary lymphoid tissue (Gunn, M. D., et al. The Journal ofexperimental medicine 1999). To monitor migration, splenic B and Tlymphocytes are purified from WASp-stabilizing SMC or control pretreatedmice that were subjected to chemotherapy or radiotherapy. Migrationtowards the chemokines SDFα and CXCL13 is induced, and measured usingthe Transwell system as previously described (Snapper S. B et al.Journal of leukocyte biology 2005). The effect of WASp-stabilizing SMCon T cell response to CCL19 and CCL21 is also measured. Although chemo-and radiotherapy destructive effects are mainly manifested byrapidly-dividing cells, increasing mature leukocyte migration and homingby WASp-stabilizing SMC is expected to improve clinical outcome ofchemo- and radiotherapy related secondary immunodeficiencies. Monocytesexit the circulation to enter inflamed tissues, where they candifferentiate into macrophages or DCs. Impaired migration of DCs tolymphoid tissues delays priming and activation of antigen-specific Tcells. Monocytes, macrophages and DCs from WAS patients are alldefective in their ability to polarize and migrate in response toinflammatory chemokines (Altman, L. C., et al. The Journal of clinicalinvestigation, 1974). The ability of isolated primary human monocytes,macrophages and DCs to polarize and migrate in response to inflammatorychemokines in vitro is examined using a standard migration assayfollowing SMC treatment (Snapper S. B et al. Journal of leukocytebiology 2005). Treatment with WASp-stabilizing SMC should enhance theability of these cells to efficiently migrate in response to externalstimuli.

Next, the in vivo ability of murine DCs to migrate and to localize tothe spleen or lymph node T cell areas is determined as previouslydescribed (Snapper S. B et al. Journal of leukocyte biology 2005). Thisis detected using the chemotherapy or radiotherapy treated mouse models,as described above. Spleen and lymph nodes are harvested, and therelative numbers of DCs are monitored using FACS analysis.

Contact of migrating myeloid cells with their substrate is mediated bypodosomes, which are actin-rich structures that are surrounded by a ringof integrins and integrin-associated proteins. Podosomes play a crucialrole in cellular motility and invasion. These structures are localizedin close proximity with the leading edge of migrating cells, and theirformation and function are dependent on WASp activity (Linder, S. &Kopp, P. Journal of cell science 2005). The ability of WASp-stabilizingSMC to increase podosome formation in macrophages and DCs is thusanalyzed by isolating macrophages and DCs from human peripheral bloodfollowed by their seeding over stimulating surfaces that promotemigration. The formation of podosomes is quantified using cell stainingwith phalloidin, which probes F-actin within the podosomes, followed byconfocal microscope analysis, as previously described (Linder, S. et al.PNAS 1999). Higher podosome formation are expected to be detectedfollowing treatment of the WASp-stabilizing SMCs vs. control.

Reverse Effector Cell Dysfunctions Using WASp-Stabilizing SMCs UponSecondary Immunodeficiency Mediated by Chemo- or Radiotherapy.

The main consequence of secondary immunodeficiency caused bychemotherapy or radiotherapy in T cells is impaired cytokine productionin response to TCR activation. WASp-deficient T cells also fail toproduce cytokines upon TCR activation (Zhang, J., et al. The Journal ofexperimental medicine, 1999). The secretion of IL-2, IFN-γ, TNF-α, IL-4,and IL-10 by activated T cells obtained from immunocompromised mice (bychemo or radiotherapy) pretreated with IV injected WASp-stabilizing SMCor control is examined.

Several reports demonstrated that chemo- and radiotherapies decreasesthe cytotolytic activity of NK cells, exposing patients to lifethreatening infections. To evaluate whether upregulation of WASpexpression rescues NK cell function in secondary immunodeficiency, NKcell cytotoxicity is analyzed in vitro in response to WASp-stabilizingSMC or control. Next, this activity is measured in vivo, inWASp-stabilizing SMC treated or control mice subjected to chemotherapyand/or radiotherapy. To examine the effect of WASp upregulation on NKcell cytotoxicity, the ability of NK cells to lyse susceptible targetcells is tested using the standard 35S release assay Matalon, O., et al.Science signaling 2016). Due to the critical role of WASp in NK cellcytotoxicity, and the negative effects of chemo- and radiotherapies onNK cell effector function, treatment with WASp-stabilizing SMCs isexpected to enhance NK cytolytic activity and to mitigate secondaryimmunodeficiencies.

Phagocytosis of pathogens and their presentation by DCs and macrophagesis an essential step in the activation of T cells and in mediatingadaptive immunity. WASp plays a key role in phagocytosis by DCs andmacrophages. Phagocytic cups enclose extracellular particles andsubsequently internalize them into phagosomes. To evaluate whetherupregulation of WASp expression rescues macrophages or DC functionsfollowing chemotherapy or radiotherapy, phagocytic activity of bonemarrow macrophages or DCs is examined. These cells are isolated formWASp-stabilizing SMC treated mice that have undergone chemotherapy orradiotherapy, and are exposed to fluorescent latex beads for 15 minutes.Following fixation, cells are labeled with cholera toxin and phalloidinto visualize the plasma membrane and cell area (by F-actin),respectively. Internalized beads are identified by single confocalplanes and 3D reconstruction of z-stacks. For quantification of uptake,only those cells that contain a bead surrounded by plasma membrane arescored as positive.

To determine the role of WASp upregulation on phagocytic activity usinga more physiological antigen, macrophages or DCs are incubated withGFP-expressing Salmonella Typhimurium, and phagocytosis will bemonitored by FACS analysis of CD11c+/GFP+ cells.

WASp is fundamental for B cell development and function in both humansand mice. Chemotherapy or radiotherapy damage the B-cell follicle, aswell as the marginal zone (MZ) architecture. To understand whether WASpupregulation may reverse this damage, immunohistochemistry of mousespleen sections is performed following IV injections of theWASp-stabilizing SMCs of the invention vs. control. Spleens are removedfrom the mice and fixed. Cryostat sections are prepared andimmunostained with anti-WASp and anti-B220, which serves as a marker forMZ B-cells to monitor MZ integrity. A normal architecture of MZ isexpected following the WASp-stabilizing SMCs of the invention.

The number of regulatory T (Treg) cells, their suppressive function andtheir homing are known to be defective in WASp-deficient hosts,indicating the key role of WASp in regulating Treg activation (Zhang,J., et al. The Journal of experimental medicine, 1999). To investigatethe effect of WASp-stabilizing SMC on the suppressive ability of Tregcells, in vitro suppression assays is performed. Briefly,CD4+CD25highCD127−/low Treg cells and CD4+CD25− effector T cells areisolated by FACS sorting from PBMCs pretreated with eitherWASp-stabilizing SMC or control. CD4+CD25− effector T cells arestimulated by CD3-depleted APCs and 1 g/ml of soluble anti-CD3 mAbs.Suppressive activity of naïve Treg (nTreg) cells is assessed byco-culture of effector T cells with nTreg cells at a 1 to 1 ratio.Proliferation of the CD4+CD25− effector T cells are evaluated by3H-thymidine incorporation or XTT assay following stimulation.Furthermore, IFNγ and IL-2 secretion by the effector T cells is measuredusing ELISA. Less proliferation of effector T cells as well as reducedsecretion of IFNγ and IL-2 by these cells upon restoration of Tregsuppressive function is expected.

Example 8 Boosting Immune Protection Against Pathogens

By improving the ability of the immune system to deal with pathogenicchallenges, WASp-stabilizing SMC offers a promising approach forimproving health outcomes. In the following experiments, the ability ofWASp-stabilizing SMC to improve key immune activities that are essentialfor eliminating infectious agents is determined. The ability tophagocytose pathogens is a key property of the innate immune response.Phagocytosis not only serves as a direct mechanism for pathogenclearance, but also provides an essential step in presenting foreignantigens to the adaptive arm of the immune system. As described above,phagocytosis is mediated by the formation of actin-based membraneinvaginations, called phagocytic cups. Since, WASp is essential for theformation of these structures, the effect of WASp-stabilizing SMC isanalyzed on monocyte uptake of FITC-labelled Escherichia coli. Briefly,isolated primary monocytes are treated with WASp-stabilizing SMC vs.control treatment, incubated with fluorescently-labelled bacteria, andFACS analysis of CD11c+/FITC+ cell (active phagocytes) percentage isconducted to monitor phagocytosis.

Additionally, the ability of macrophages and DCs to phagocytoseapoptotic cells and latex beads is examined. The phagocytic cells areincubated with fluorescent latex beads, fixed, and fluorescently labeledusing cholera toxin and phalloidin. Quantification of internalized beadsis performed using confocal microscopy, as described above.

Antibody production by B cells is a key component of the immune responseto bacterial infections. WASp plays a pivotal role in the primaryhumoral immune response. To evaluate the antibody response to variousantigens expressed on different types of pathogens, in vivo assays usingantigens of different origins are performed including viral (e.g. CMV,EBV, influenza), or bacterial antigens (e.g. Mycobacterium tuberculosis,MTB; Streptococcus pneumoniae, pneumococcus or S. pneumoniae). To thisend, C57BL/6 mice are inoculated with pathogenic antigens followed bytreatment with WASp-stabilizing-SMC or control. Mice are challengedintraperitoneally (I.P) with 0.5 μg/mouse of a vaccine containing amixture of highly purified capsular polysaccharides from the most 23prevalent or invasive pneumococcal strains of Streptococcus pneumoniae(Pneumovax23; commercially available (Pneumovax23); Sanofi Pasteur MSD,Lyon, France. After 7 days, blood samples are collected from the facialvein of mice, and serum IgM antibodies specific for Pneumovax23polysaccharidic components are measured by ELISA. Higher IgM levels inthe serum of mice treated with the WASp-stabilizing SMC are expected dueto enhanced B cell activation.

Example 9 Determine the Therapeutic Potential of WASp-Stabilizing SMCfor Treating Thrombocytopenia and Specifically, IdiopathicThrombocytopenic Purpura (ITP)

ITP is an autoimmune disease characterized by microthrombocytopeniacaused by platelet clearance due to production of auto-antibodiestargeting platelets. Since the expression of WASp is crucial forplatelet production from megakaryocytes, the effect of WASp-stabilizingSMC on platelet activity and on the thrombocytopenic phenotype in ITPmouse models is examined.

First, the effect of WASp-stabilizing SMC on platelet function in vitrois determined. Platelets are isolated from the blood of healthy donorsand incubated with WASp-stabilizing SMC in comparison to control treatedcells. Adhesion of platelets to fibrinogen is a key process in plateletaggregation, mediated by integrins, such as αIIbβ3. WASp is importantfor αIIbβ3-mediated-cell adhesion of platelets and megakaryocytes.Several clinical observations indicate that WASp deficiency causes asubstantial decrease in platelet number and aggregation. This leads toheavy bleeding in WAS/XLT patients. Thus, the binding of FITC-labelledsoluble fibrinogen to platelet integrin αIIbβ3 following adenosinediphosphate (ADP) stimulation is determined via FACS analysis. Thetreatment with WASp-stabilizing SMC is expected to enhance plateletaggregation, relative to control cells in addition to the plateletactivation demonstrated in FIG. 11.

F-actin polymerization and rearrangement is pivotal for plateletactivation and function. F-actin content per platelet is measured byquantitating FITC phalloidin staining using FACS analysis following ADPstimulation. Enhanced F-actin content per platelet is anticipated inresponse to treatment with the WASp-stabilizing SMCs of the invention,which is consistent with the data of FIG. 5 and FIG. 6 demonstratingupregulated WASp expression upon platelet treatment with the same SMC.

Platelet aggregation is mediated mainly by the activity of αIIbβ3integrin, which undergoes a conformational change following plateletactivation. The conformation of αIIbβ3 is converted from a low-affinitystate to a high-affinity state following agonist stimulation. WASplevels, which are reduced in WAS/XLT patients, are not sufficient toassociate the actin cytoskeleton to αIIbβ3 sites or to optimize theaffinity of αIIbβ3 for its ligand. Thus, the ‘affinity-maturation’ ofαIIbβ3 to its ligand following agonist stimulation is impaired in thesepatients. In order to determine the effect of WASp expression on αIIbβ3activity, platelets are treated with WASp-stabilizing SMC, stimulatedwith thrombin (αIIbβ3 ligand), and stained with fluorescently labelledPAC-1 antibody. This monoclonal antibody recognizes only the activatedform of αIIbβ3 extracellular domain. The activation of the αIIbβ3integrin is measured by FACS analysis, assuming increased integrinactivation following treatment with the WASp-stabilizing SMCs of theinvention.

Following their activation and F-actin polymerization, platelets mustspread over intact blood vessels in the process of clot formation.Platelets are treated with WASp-stabilizing SMC followed by seeding overimmobilized fibrinogen-coated slides. Spreading is analyzed as follows:platelets are stained with the fluorescent label calcein-AM, after theirseeding over immobilized fibrinogen-coated 96 plates. Fluorescent signalis measured by fluorescence spectrophotometry after extensive washing ofwells, to determine platelet adhesion as an indication of theirspreading capacity.

Microthrombocytopenia is characterized by reduced expression of plateletsurface markers, including GPIb, GPV, GPIX, CD9, GPVI, αIIbβ3. Theexpression of these surface proteins is determined in platelets isolatedfrom the ITP mouse model, (NZW x BXSB) F1 mouse (also known as W/B F1mice), following IV injection with WASp-stabilizing SMC vs. control.Cells are stained with specific antibodies for the indicated surfaceproteins followed by FACS analysis. The expression of these markers isexpected to be increased following SMC treatment.

Platelets obtained from WAS/XLT patients are characterized by aberrantmorphology and size resulting in reduced mean platelet volume. Since lowmean platelet volume and short platelet lifespan are also hallmarks ofITP, these parameters are examined in W/B F1 mice treated withWASp-stabilizing SMC vs. control. Clearance of platelets from the bloodcirculation (reflecting platelet lifespan) is determined by theretro-orbital injection of fluorescently-labelled antibody against GPIX,a unique surface marker expressed over all platelets, into male W/B F1mice. The percentage of labelled platelets is determined by daily bloodwithdrawal and subsequent FACS analysis. In addition, platelet volume isdetermined with an automated blood cell analyzer. Both platelet lifespan and volume are expected to increase following SMC-stabilizing WASptreatment. The microthrombocytopenia in XLT patients is caused by bothaccelerated platelet clearance by macrophages and the premature releaseof platelets from their precursors into the BM due to alteredcytoskeletal dynamics. Thus, platelet release from megakaryocytes ismeasured by immunofluorescence staining of BM-cross sections. Stainingwith anti-GPIb (for platelet and megakaryocyte detection) and anti-CD105(for endothelium detection) is performed, and analyzed by confocalmicroscopy. To this end, W/B F1 mice are utilized followingWASp-stabilizing SMC vs. control treatment to determine whetherincreased WASp expression enhances platelet count.

To confirm that WASp-stabilizing SMC present potential therapeuticapproach for ITP by improving platelet count and size, theaforementioned experimental procedures are also conducted on bloodsamples from human ITP patients.

Example 10 Supportive Treatment for the Rapid Reconstitution ofLeukocyte and Megakaryocyte Populations Following HSCT, Gene Therapy orAdoptive Cell Transfer

Successful human stem cell transplantation (HSCT) is a challengingprocedure that is highly dependent on the ability of thedonor/autologous stem cells to accommodate the recipientmicroenvironment. Enhancement of WASp expression restores hematopoieticcell growth and survival, which is a key factor for successfulreconstitution of the host system. Thus the ability of WASp-stabilizingSMC to improve BM engraftment and hematopoietic reconstitution isdetermined. To this end, recipient mice are subjected to eitherWASp-stabilizing SMC or control treatment. Syngeneic B6D2F1 male miceserve as donors for matched female bone marrow recipients. Bone marrowtransplantation is performed as follows: briefly, following whole bodyirradiation with 1,200 cGy, the female recipients are injected via thetail vein with 20,000 male whole BM cells.

The reconstitution of the hematopoietic system is examined by bloodcollection from mice, followed by FACS analysis with gating on thefollowing population: T (CD3+), NK (CD3-CD56+) and B (CD19+) cells usingFACS. The relative distribution of cell subsets is analyzed to confirmnormal reconstitution of lymphoid cells following WASp-stabilizing SMCtreatment.

To further examine hematopoietic system reconstitution, tissue sectionsof liver and spleen is formalin-fixed and paraffin embedded. To detectdonor-derived cells in these tissues, fluorescence in situ hybridization(FISH) for the Y-chromosome is performed. Male BM engraftment as well asBM-derived cells are quantified by FISH. Intense Y-chromosome probestaining in sections obtained from the mice treated withWASp-stabilizing agents is expected.

Example 11 Upregulation of Anti-Cancer Immune Response by BoostingCytotoxic Lymphocyte Activity

NK cells and CTLs constitute the two major cytotoxic effector immunepopulations, able to directly mediate lysis of viral infected andcancerous cells. WASp deficiency, either in mice or in WAS patients,results in compromised activation and function of cytotoxic lymphocytes,impairing tumorsurveillance. In accordance with this defectivecytotoxicity, WAS patients demonstrate increased frequency ofmalignancies, with 13% of WAS patients developing malignancy at anaverage age of 9.5 years. Thus, the role of WASp-stabilizing SMC inenhancing NK and CTL activation and cytotoxicity is determined.Following formation of a stable effector-cancer cell conjugates,cytotoxic effector cells must first elevate their intracellular Ca²⁺flux to achieve activation. Intracellular calcium concentration ismeasured upon NK cell interaction with 721.221 B lymphoma target cellsfollowing NK cell incubation with WASp-stabilizing SMC. For thispurpose, the human NK YTS line, or primary isolated cells is used. Asshown in FIG. 11, WASp-stabilizing SMCs increase calcium flux inplatelets upon activation. Similar effects in NK cells are expected.

Next, the role of WASp-stabilizing SMC in enhancing NK cell effectorfunctions i.e. cytotoxicity and/or cytokine production is examined. Toaddress this goal, the effects of WASp-stabilizing SMC are studied onprimary or YTS NK cells interacting with the 721.221 cancer cell line.The ability of NK cells to mediate target cell cytotoxicity is measuredusing 35S-release (Lee, S. H., et al. J Immunol 2009) and degranulationanalyses (Anfossi, N., et al. Immunity 2006). IFNγ secretion by the NKcells following WASp-stabilizing SMC and target cell incubation ismeasured as well by ELISA. Increases in all NK cell functions areexpected following WASp upregulation.

NK cells play a major role in the elimination of metastatic cells andsmall tumor grafts, and exhibit an antitumor response in patients afterleukemia relapse. Therefore, the effect of WASp-stabilizing-SMC on NKcell killing efficiencies of different cancerous cell targets isdetermined. For this purpose, the following human cancer cell lines areused as target cells: 1106mel melanoma cells, HeLa cervical carcinoma,and A549 and H1975 Lung Carcinoma cell lines. The killing efficiency ofthese cancer cells is measured using the 35S-release assay (Lee, S. H.,et al. J Immunol 2009) following NK cell pretreatment withWASp-stabilizing SMC vs. control.

To extend these experiments, NK cell killing efficiency of variousprimary patient-derived tumor cells is determined, using the abovedescribed experimental system. In addition, the anti-tumor effect ofhuman NK cells is examined in vivo using humanized SCID mice carryinghuman tumor xenografts.

Example 12

The SMCs of the Invention in Combined Supportive Treatment with G-CSF

It is important to emphasize that few agents exist that are able toboost the immune response, and those that are under development are atvery early stages. Almost no suitable agents are available, with theexception of granulocyte colony stimulating factor (G-CSF). Currently,modalities for boosting the immune response are limited to lifestylechanges e.g. physical activity and diet.

G-CSF is a growth factor that stimulates the production, maturation, andactivation of neutrophils. GCSF stimulates the release of neutrophilsfrom the bone marrow. It is being used for patients receivingchemotherapy to accelerate the recovery of neutrophils, reducing theneutropenic phase. Today, hundreds of thousands of patients are treatedwith G-CSF to boost their immune systems after chemotherapy or BMtransplants. G-CSF may also be used after a stem cell transplant to helpaccelerate recovery of the new stem cells in the bone marrow.

While G-CSF exclusively induces differentiation of granulocytes fromhematopoietic progenitor cells, WASp is known to enhance the activity ofall immune cells and platelets. Furthermore, WASp was widelydemonstrated to increase the growth rate and survival of maturelymphocytes including T, B, NK and Treg. Given the multipleimmunological tasks coordinated by WASp through its regulation of theactin cytoskeleton, and specifically, actin-dependent processes,pharmacological intervention in WASp-mediated signaling pathways isexpected to have greater effects on the immune system than GCSF, whichaffects a single proliferative process. Therefore, WASp-stabilizing SMCscould eventually evolve as a rational strategy for the treatment ofsecondary immunodeficiencies. In contrast to G-CSF, the major activityof WASp is on differentiated or mature hematopoietic cells. By acting ondifferent processes necessary for an appropriate and robust immuneresponse, administration of G-CSF and the WASp-stabilizing SMCs might becomplimentary approaches with additive value. However, administration ofWASp-stabilizing SMCs may be relevant in cases in which G-CSF will nothave an effect e.g. immune dysfunction of the lymphatic system.

To assess whether the WASp-stabilizing-SMCs of the invention could beused in a combined treatment regimen with other supportive agents, inrestoring normal lymphoid cell counts, mice are subjected tochemotherapy or radiotherapy and hematopoietic recovery is monitored. Todetermine the effect of the combined SMC and G-CSF treatment on theimmune response following chemotherapy, C57BL/6 mice are treated withthe chemotherapeutic agents oxaliplatin (5 mg/kg), or doxorubicin (2 mMin 100 ml PBS) injected intraperitoneally (IP).

For radiotherapy experiments, C57BL/6 mice are briefly anesthetizedusing isoflurane, and subjected to total body X-irradiation at a dose of7.5 Gray (Gy). Mice are used at 6 to 20 weeks of age. The mice areinjected intravenously (IV) with the WASp-stabilizing SMCs of theinvention vs. control and monitored by complete blood cell counts todetermine the absolute numbers of T, B, and NK cells. Blood sample arewithdrawn from the tail vein and the cells are counted primarily using ablood analyzer (KX21N, Sysmex, Kobe, Japan) followed by Giemsa-stainingof blood smears.

Additionally, total cell counts in bone marrow, thymus, and spleen areevaluated after preparation of single-cell suspensions and eliminationof red blood cells.

1-50. (canceled)
 51. A method for modulating degradation and/orstabilizing of WASp in a cell, comprising the step of contacting saidcell with an effective amount of at least one SMC modulator of WASpdegradation, or any vehicle, matrix, nano- or micro-particle or anycomposition comprising the same, said SMC modulator having the generalformula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof, wherein R₁ and R₂ areeach independently from each other selected from H, straight C₁-C₁₂alkyl, a ring system containing five to seven atoms, each optionallysubstituted by at least one a ring system containing five to seven atomsoptionally substituted by at least one halide or straight C₁-C₅ alkyl orR₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl, L1 and L2 if present are each independently fromeach other selected to be absent or from —CH₂—(CH₂—C(O)—N)—(CH₂)₂,—(CH₂)—S—, —(CH₂)—, —(CH₂)—O—, —NH—(CH₂)—, and each optionallysubstituted with C₁-C₅ alkyl, a ring system containing five to twelveatoms optionally substituted with C₁-C₅ alkyl; R₃ and R₄ are eachindependently from each other absent or selected from a ring systemcontaining five to 12 atoms, each optionally substituted with at leastone of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅, R₅ is an aring system containing five to seven atoms optionally substituted by atleast one halide or straight C₁-C₅ alkyl.
 52. The method according toclaim 51, wherein said SMC modulator is having the general formula(XII):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof, wherein R₁ and R₂ areeach independently from each other selected from H, straight C₁-C₁₂alkyl, a ring system containing five to seven atoms, each optionallysubstituted by at least one a ring system containing five to seven atomsoptionally substituted by at least one halide or straight C₁-C₅ alkyl;or R₁ and R₂ together with the nitrogen atom they are connected to forma five to seven membered unsaturated ring optionally include at leastone of N, O and optionally substituted with at least one of straightC₁-C₅ alkyl, L1 and L2 are absent or each independently from each otherselected from —(CH₂)_(n)—, (CH₂)_(n)—S—, —(CH₂)_(n)—O—, and may beoptionally substituted with C₁-C₅ alkyl, R3 is a ring system containingfive atoms, optionally substituted with at least one straight C₁-C₅alkyl, or R₅, R₅ is an a ring system containing five to seven atomsoptionally substituted by at least one halide or straight C₁-C₅ alkyl,R4 is absent or selected from a ring system containing five to 12 atoms,each optionally substituted with at least one of straight C₁-C₅ alkyl.53. The method according to claim 52, wherein said SMC modulator is oneof:

1-(2,6-Dimethyl-piperidin-1-yl)-2-[5-(quinolin-8-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-ethanone(designated herein as SMC 34) or

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-3-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(designated herein as SMC 34.7).
 54. The method according to claim 51,wherein said SMC modulator is having the general formula (XIII):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof, wherein R₁ and R₂ areeach independently from each other selected from H, a ring systemcontaining five to seven atoms, each optionally substituted by at leastone of halide, amide, nitro; L1 and L2 if present are each independentlymay be absent or from each other selected from —CH₂—(CH₂—C(O)—N)—(CH₂)₂,—(CH₂)—S—, —(CH₂)—, —(CH₂)—O—, —NH—(CH₂)— each independently from eachother optionally substituted with C₁-C₅ alkyl, a ring system containingfive to seven atoms optionally substituted with C₃-C₅ alkyl; R₃ and R₄are each independently from each other absent or selected from a ringsystem containing five to 12 atoms, each optionally substituted with atleast one of straight C₁-C₅ alkyl, (═O), (═S), or R₅, R₅ is an a ringsystem containing five to seven atoms optionally substituted by at leastone halide or straight C₁-C₅ alkyl.
 55. The method according to claim54, wherein said SMC modulator is one of:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide(Designated herein as SMC#33); or

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester (Designated herein as SMC#30).
 56. The methodaccording to claim 51, wherein said modulation results in reduced WASpdegradation in a cell, and wherein said cell is a non-erythroidhematopoietic cell.
 57. The method according to claim 51, wherein saidcell is of a subject suffering from an innate or acquired immune-relateddisorder or condition.
 58. A method for treating, preventing,inhibiting, reducing, eliminating, protecting or delaying the onset of aan innate or acquired immune-related disorder or condition in a subjectin need thereof, said method comprises administering to said subject atherapeutically effective amount of at least one SMC modulator of WASphaving the general formula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof, wherein R₁ and R₂ areeach independently from each other selected from H, straight C₁-C₁₂alkyl, a ring system containing five to seven atoms, each optionallysubstituted by at least one a ring system containing five to seven atomsoptionally substituted by at least one halide or straight C₁-C₅ alkyl orR₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl, L1 and L2 if present are each independently fromeach other selected to be absent or from —CH₂—(CH₂—C(O)—N)—(CH₂)₂,—(CH₂)—S—, —(CH₂)—, —(CH₂)—O—, —NH—(CH₂)—, and each optionallysubstituted with C₁-C₅ alkyl, a ring system containing five to twelveatoms optionally substituted with C₁-C₅ alkyl; R₃ and R₄ are eachindependently from each other absent or selected from a ring systemcontaining five to 12 atoms, each optionally substituted with at leastone of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅, R₅ is an aring system containing five to seven atoms optionally substituted by atleast one halide or straight C₁-C₅ alkyl.
 59. The method according toclaim 58, wherein said SMC modulator is one of:

1-(2,6-Dimethyl-piperidin-1-yl)-2-[5-(quinolin-8-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-ethanone(designated herein as SMC 34); or

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-3-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(designated herein as SMC 34.7).
 60. The method according to claim 58,wherein said SMC modulator is one of:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide(Designated herein as SMC#33); or

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester (Designated herein as SMC#30).
 61. The methodaccording to claim 58, wherein said immune-related disorder or conditionis a primary or a secondary immunodeficiency.
 62. The method accordingto claim 61, wherein said primary immunodeficiency is a hereditary oracquired disorder associated with WASp dysfunction and wherein saidhereditary disorder associated with WASp dysfunction is at least one ofWAS and XLT, or any condition or disorder associated therewith.
 63. Themethod according to claim 61, wherein said secondary immunodeficiency iscaused by at least one of chemotherapy, radiotherapy, biologicaltherapy, bone marrow transplantation, gene therapy, adoptive celltransfer or any combinations thereof.
 64. The method according to claim58, wherein said immune-related disorder or condition is a pathologiccondition caused by at least one pathogen.
 65. The method according toclaim 58, wherein said immune-related disorder or condition isthrombocytopenia.
 66. The method according to claim 58, wherein saidimmune-related disorder or condition is cancer.
 67. The method accordingto claim 66, wherein said subject undergoes at least one ofchemotherapy, radiotherapy, biological therapy, or any combinationsthereof, and wherein said method further comprises the step ofadministering to said subject before, simultaneously with, after or anycombination thereof, the administration of said SMC modulator, at leastone agent that induces differentiation of hematopoietic progenitorcells.
 68. A SMC modulator of WASp degradation having the generalformula (XI):

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer orphysiologically functional derivative thereof, or a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone of said SMC and optionally, at least one pharmaceutically acceptablecarrier/s, excipient/s, auxiliaries, and/or diluent/s; wherein R₁ and R₂are each independently from each other selected from H, straight C₁-C₁₂alkyl, a ring system containing five to seven atoms, each optionallysubstituted by at least one a ring system containing five to seven atomsoptionally substituted by at least one halide or straight C₁-C₅ alkyl orR₁ and R₂ together with the nitrogen atom they are connected to form afive to seven membered saturated or unsaturated ring optionally includeat least one of N, O and optionally substituted with at least one ofstraight C₁-C₅ alkyl, L1 and L2 if present are each independently fromeach other selected to be absent or from —CH₂—(CH₂—C(O)—N)—(CH₂)₂,—(CH₂)—S—, —(CH₂)—, —(CH₂)—O—, —NH—(CH₂)—, and each optionallysubstituted with C₁-C₅ alkyl, a ring system containing five to twelveatoms optionally substituted with C₁-C₅ alkyl; R₃ and R₄ are eachindependently from each other absent or selected from a ring systemcontaining five to 12 atoms, each optionally substituted with at leastone of straight or branched C₁-C₅ alkyl, (═O), (═S) or R₅, R₅ is an aring system containing five to seven atoms optionally substituted by atleast one halide or straight C₁-C₅ alkyl.
 69. The SMC modulatoraccording to claim 68, being one of: I. the SMC being one of:

1-(2,6-Dimethyl-piperidin-1-yl)-2-[5-(quinolin-8-yloxymethyl)-[1,3,4]oxadiazol-2-ylsulfanyl]-ethanone(designated herein as SMC 34); or

1-(2,6-Dimethyl-piperidin-1-yl)-2-(4-methyl-5-pyridin-3-yl-4H-[1,2,4]triazol-3-ylsulfanyl)-ethanone(designated herein as SMC 34.7); or II. the SMC being one of:

N′-[2-(benzylamino)-2-oxoethyl]-N′-(2-methylphenyl)-N-(1,3-thiazol-2-yl)butanediamide(Designated herein as SMC#33); or

3-(4-Cyclopentylcarbamoyl-benzyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydro-quinazoline-7-carboxylicacid methyl ester (Designated herein as SMC#30).
 70. A kit comprising:(a) at least one SMC modulator of WASp according to claim 68; and atleast one of: (b) at least one chemotherapeutic agent; (c) at least onebiological therapy agent; and (d) at least one agent that inducesdifferentiation of hematopoietic progenitor cells.